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玻璃体腔注射康柏西普对黄斑水肿患者角膜厚度和内皮细胞的影响王华;席亚慧;沈兰珂;贺春香【摘要】目的:研究玻璃体腔注射康柏西普对黄斑水肿患者角膜厚度及内皮细胞的影响.方法:选取2017-01/12在本院行玻璃体腔注射康柏西普治疗黄斑水肿的患者30例30眼,手术前后使用超声生物显微镜测量中央角膜厚度,使用角膜内皮计数仪测量患眼中央角膜内皮细胞密度和六角形细胞比例.结果:本组患者术前和术后1d,1wk角膜中央厚度分别为551.68±12.80、552.06±13.22、552.49±13.83μm(P>0.05).术前和术后1d,1wk,3、6mo中央角膜内皮细胞密度分别为2551.03±287.55、2563.79±292.34、2543.32±282.41、2526.18±280.24、2519.60±279.89个/mm2,六角形细胞比例分别为(50.23±7.51)％、(50.93±8.23)％、(50.60±7.91)％、(50.40±7.50)％、(50.93±8.19)％,均无明显差异(P>0.05).结论:玻璃体腔注射康柏西普是一种安全的治疗黄斑水肿的方法,在注射后6mo内对角膜厚度及内皮细胞无明显影响.【期刊名称】《国际眼科杂志》【年(卷),期】2019(019)004【总页数】3页(P657-659)【关键词】康柏西普;角膜厚度;角膜内皮细胞【作者】王华;席亚慧;沈兰珂;贺春香【作者单位】725000 中国陕西省安康市中医医院眼科;725000 中国陕西省安康市中医医院眼科;725000 中国陕西省安康市中医医院眼科;725000 中国陕西省安康市中医医院眼科【正文语种】中文0引言玻璃体腔注射抗血管内皮生长因子(VEGF)药物可有效抑制黄斑区新生血管的生长。
专利名称：APPLICATION OF MESENCHYMAL STEMCELLS IN PROPHYLAXIS OR TREATMENT OFSTRESS RESPONSE-INDUCED WEAKENEDIMMUNITY发明人：SHI, Yufang,CAO, Gang申请号：EP14795387.1申请日：20140429公开号：EP2997971A4公开日：20170607专利内容由知识产权出版社提供摘要：The present invention discloses the application of mesenchymal stem cells in the prophylaxis or treatment of stress response-induced weakened immunity. More particularly, the present invention provides the use of mesenchymal stem cells in preparing a pharmaceutical composition for the prophylaxis or treatment of stress response-induced lymphopenia; and/or the use thereof in preparing a pharmaceutical composition for the prophylaxis or treatment of stress response-induced weakened immunity; and/or the use thereof in preparing a pharmaceutical composition for the promotion of interleukin 4 (IL-4) expression and/or enhanced or strengthened activity. The present invention has found that mesenchymal stem cells can, by means of regulating IL-4 and down-stream related signaling pathways, protect against hormonal disorder-induced lymphopenia, thus having the function of activating immune response.申请人：Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences 地址：319 Yueyang Road Xuhui District Shanghai 200031 CN国籍：CN代理机构：dompatent von Kreisler Selting Werner - Partnerschaft von Patent- und Rechtsanwälten mbB更多信息请下载全文后查看。
简介杨梦甦教授1984年毕业于厦门大学化学系，1993年获得加拿大多伦多大学博士学位，1993-1994期间在美国加州生物医学机构Scripps Research Institute从事研究工作。
1994年底加入香港城市大学，现任香港城市大学生物及化学系讲座教授和香港城市大学深圳生物医药中心主任，并兼任英国中兰开夏大学、浙江大学、解放军第三军医大学、沈阳药科大学、及解放军总医院客座教授。
在行政工作方面担任香港城市大学校董会荣誉学位委员会、大学顾问委员会、大学教务会、研究生院理事会等多个委员会成员。
杨教授主要研究领域为生物芯片技术的开发与应用和纳米生物领域的基础与应用研究, 1995年迄今成功获得包括国家高科技发展计划（863计划）、香港特区政府创新科技基金和研究资助局、及香港赛马会基金等逾6千万港元研究基金的资助，负责管理多个大型科研项目，亦是香港特区政府大学资助委员会之“药物发现与合成分子技术研究”、“中药基础研究与开发”、“海洋环境研究与创新技术”三个卓越学科的主要成员之一。
迄今在国际权威杂志共发表论文160余篇，专著章节17篇，在国内核心杂志发表论文50余篇，在大型国际会议共发表论文80余篇，应邀在各大学和研究机构做过逾70场学术报告，在相关领域申请/获得20多项中国及美国专利。
杨教授实验室已培训18名博士、8名硕士及16名博士后。
杨教授为香港特区政府创新科技基金评审委员会和健康防护基金评审委员会成员，及香港特区政府创新科技署纳米技术和先进材料研究院(NAMI)技术顾问，并兼任英国皇家化学会专业杂志《The Analyst》和中国化学会专业杂志《生命科学仪器》编辑委员会成员，德国《Microchimica Acta》国际顾问委员会成员，以及国际生物芯片大会学术委员会和世界华人高科技化学大会学术委员会成员。
杨教授从2002年主持筹建香港城市大学深圳生物医药中心，领导该中心成功获得包括国家高科技发展计划（863）课题及广东省科技重点攻关项目的资助，并得到深圳市/广东省政府的多次奖励，被授予深圳市生物芯片重点实验室的称号。
MSC治疗小鼠OB模型排斥反应的作用研究史乾;李静;范慧敏【摘要】目的：研究间充质干细胞(MSC)免疫抑制功能在治疗小鼠闭塞性细支气管炎(OB)中的作用。
方法分离C57BL/6小鼠骨髓MSC,建立小鼠气管移植后OB 反应模型,移植当天给予MSC,30 d后处死小鼠,移植气管HE染色,观察移植气管急性排斥反应的发生情况。
并用酶联免疫吸附试验(ELISA)检测移植排斥相关炎症因子的含量。
结果 MSC治疗移植组与异系移植组相比,排斥反应得到缓解,与无排斥反应的同系移植对照组相似,移植气管管腔阻塞程度缓解。
移植气管ELISA检测发现, MSC治疗移植组小鼠体内炎症因子IFN-g下降,而抑炎因子白细胞介素-10(IL-10)上升。
结论 MSC能缓解小鼠气管移植后OB反应,并调节体内免疫状态,抑制炎症。
%Objective To analyze the effect of immunosuppression of mesenchymal stem cell (MSC) in the treatment of mice obliterans bronchiolitis (OB). Methods Marrow MSC of C57BL/6 mice was separated to set OB reaction model after trachea transplantation of mice. MSC was given on the day of transplantation. The mice were put to death after 30 days. HE staining was used on the transplanted trachea, and the situation of acute rejection of transplanted trachea was observed. Enzyme-linked immuno sorbent assay (ELISA) was applied to detect the content of inflammatory factors related to transplant rejection. Results Rejection reaction in MSC transplant group was eased, compared with different system transplant group, and degree of lumen obstruction in transplanted trachea was also eased, compared with allografts transplant control group without rejection. ELISA test for transplanted trachea showed thatinflammatory factor IFN-g decreased in MSC transplant group, while anti-inflammatory factor interleukin-10 (IL-10) increased. Conclusion MSC can ease the OB reaction after trachea transplantation, adjust immune state and inhibit inflammation.【期刊名称】《中国现代药物应用》【年(卷),期】2014(000)019【总页数】2页(P1-2)【关键词】间充质干细胞;闭塞性细支气管炎;白细胞介素-10【作者】史乾;李静;范慧敏【作者单位】200120 同济大学附属上海市东方医院心外科，心力衰竭研究所;200120 同济大学附属上海市东方医院心外科，心力衰竭研究所;200120 同济大学附属上海市东方医院心外科，心力衰竭研究所【正文语种】中文肺移植和心肺联合移植是治疗多种终末期心肺疾病的重要措施, 尽管免疫抑制剂的广泛使用能较为有效的防治急性期排斥, 然而其对慢性排斥的作用却收效甚微。
细胞培养用青霉素-链霉素产品简介：细胞培养用青霉素-链霉素(Penicillin-Streptomycin for Cell Culture)为粉剂，是最常用的细胞培养用抗生素(即通常所谓的双抗)。
在细胞培养液中推荐的青霉素的工作浓度为100U/ml ，链霉素的工作浓度为0.1mg/ml 。
一个包装的细胞培养用青霉素-链霉素可以配制80L 细胞培养液。
保存条件：室温保存。
4ºC 保存可以使用更长时间。
注意事项：开瓶后需防止受潮。
本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：细胞培养用青霉素-链霉素可以参考如下两种方法之一使用：1. 配制细胞培养液时加入细胞培养用青霉素-链霉素，然后再过滤除菌：配制细胞培养液时按照青霉素的工作浓度为100U/ml ，链霉素的工作浓度为0.1mg/ml 进行配制，配制完成后过滤除菌即可使用。
2. 配制青霉素-链霉素溶液(100X)母液，然后再添加到细胞培养液中：按照青霉素的含量为10KU/ml ，链霉素的含量为10mg/ml ，配制青霉素-链霉素溶液(100X)母液。
过滤除菌后即可按照100倍稀释加入到细胞培养液中使用。
配制的母液可以-20ºC 冻存。
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BAG2 amelioratesendoplasmic reticulum stress-induced cell apoptosis in Mycobacterium tuberculosis-infected macrophages through selective autophagy.Autophagy. 2019 Nov 11:1-15.68.Yuan K,Lai C,Wei L,Feng T,Yang Q,Zhang T,Lan T,Yao Y,XiangG,Huang X. The Effect of Vascular Endothelial Growth Factor on Bone Marrow Mesenchymal Stem Cell Engraftment in Rat Fibrotic Liver upon Transplantation. Stem Cells Int. 2019 Dec 4;2019:5310202.Version 2021.11.04碧云天/Beyotime 400-1683301/800-8283301 ST488 细胞培养用青霉素-链霉素 3 / 3。
碧云天生物技术/Beyotime Biotechnology 订货热线：400-168-3301或800-8283301 订货e-mail ：****************** 技术咨询：***************** 网址：碧云天网站 微信公众号GFP 抗体(小鼠单抗)产品编号 产品名称包装 AG281GFP 抗体(小鼠单抗)>40次产品简介：来源 用途 抗体识别位点抗体类型 GFP 分子量MouseWB, IP, IFFull length GFP IgG2a~27kDWB, Western blot; IP, Immunoprecipitation; IF, Immunofluorescence. 本GFP 抗体(小鼠单抗)，即mouse monoclonal GFP antibody ，为进口分装，用全长的来源于水母的GFP (green fluorescent protein)作为抗原制备得到的抗GFP 小鼠单克隆抗体。
克隆号为B-2。
本GFP 抗体可以识别GFP以及GFP 的一些突变体例如EGFP (enhanced green fluorescent protein)。
GFP 或其突变体EGFP 等被广泛用于基因表达效率的检测，以及和目的蛋白融合表达用于检测目的蛋白的表达和分布。
本GFP 抗体不仅可以检测GFP 或其适当的突变体，也可以检测和GFP 或其适当的突变体融合表达的蛋白。
配套提供了Western 一抗稀释液，可以用于Western 检测时的一抗稀释。
)：40次。
包装清单：产品编号 产品名称包装 AG281-1 GFP 抗体(小鼠单抗) 40µl AG281-2 Western 一抗稀释液40ml —说明书1份保存条件：GFP 抗体(小鼠单抗)-20ºC 保存，Western 一抗稀释液-20ºC 或4ºC 保存，一年有效。
单细胞悬液制备⽅法汇总⼀、外周⾎样本（图⽚来源⽹络）⼈外周⾎单个核细胞（Peripheralblood mononuclear cells ，PBMCs ）的分离制备：1）医院及其⾎液中⼼采取的新鲜的肝素抗凝的2ml外周静脉⾎备⽤；2）将肝素抗凝的静脉⾎⽤等体积的PBS稀释并充分混匀；3）将⼈淋巴细胞分离液ficoll从 4 °C 取出，恢复⾄室温，取4ml转移⾄15ml 试管中备⽤；4）⽤⽆菌吸管吸取稀释后的静脉⾎，沿管壁缓慢⼊⾄⼈淋巴细胞分离液液⾯上（ficoll分离液：抗凝⾎1:1），缓缓地不要晃荡，保持界⾯的清楚；5）将加完外周静脉⾎后的 50 ml 试管放⼊台式离⼼机中，2200 rpm ⽔平离⼼，室温 25 min，注意离⼼机升降速都调⾄最慢，降速设置中⼀定要设置成no break，或者只有1成的制动。
6）离⼼完毕后，⼩⼼取出15 ml 试管，离⼼后管内可分为三层，在上、中层界⾯处有⼀层以单个核细胞为主的⽩⾊云雾层狭窄带，即为我们所需要的单个核细胞；7）⽤⽆菌吸管吸取⽩⾊云雾层狭窄带的单个核细胞，并置于另⼀ 15 ml 离⼼管中，加⼊5mlPBS，1500 rpm，室温离⼼ 5min，并充分洗涤细胞两次；8）末次离⼼后，弃去上清，加⼊完全 RPMI 1640 培养液重悬细胞，充分混匀；9）⽤⽩细胞计数液计数单个核细胞，根据实验所需，加⼊完全 1640培养液调整细胞浓度到所需要的浓度。
⼆、组织样本（图⽚来源⽹络）常见的组织类型：⼩⿏脾脏、肝脏、⼼脏、肺脏、脑组织、肿瘤组织、⼈肿瘤组织、⽪肤组织……传统组织处理⽅法：机械法：⽹搓法、研磨法适⽤样本类型：脾脏、淋巴结、胸腺⽹搓法1、将300 ⽬尼龙⽹扎在⽆菌的⼩烧杯上；2、把剪碎的组织放在⽹上，以眼科镊⼦轻轻搓组织块，边搓边加⽣理盐⽔冲洗，直到将组织搓完；3、收集细胞悬液于离⼼管中， 1500rpm离⼼5min；4、弃上清液，加红细胞裂解液重悬沉淀，室温作⽤5min ，加等体积的PBS 中和后，1500rpm 离⼼5min ，弃上清，⽤PBS 洗涤⼀次，再⽤PBS 重悬，细胞计数后即可使⽤。
不同来源间充质干细胞生物学特性差异方洪松;周建林;彭昊;邓爽;翁金清;刘丰;陈森;周观金【摘要】背景：间充质干细胞在体内或体外特定的诱导条件下，可分化为软骨、肌肉、肌腱等。
间充质干细胞进行的临床试验主要包括组织损伤修复，如骨、软骨、关节损伤的修复，心脏、肝脏、脊髓损伤和神经系统疾病的治疗。
<br> 目的：比较各种来源的间充质干细胞的生物学特性。
<br> 方法：检索1987到2015年PubMed数据库和中国知网数据库收录的与间充质干细胞来源，间充质干细胞生物学特性相关的文献。
从细胞表面标记物，增殖、分化、迁移能力，以及功能方面进行分析总结，探讨了各种来源的间充质干细胞的优缺点。
<br> 结果与结论：不同来源的间充质干细胞的增殖潜力和表面标记物存在差异。
不同组织来源的间充质干细胞免疫活性可能与间充质干细胞处于不同组织中的活化状态、种属差异、组织来源和培养条件的不同有关，从而导致不同源性间充干细胞免疫活性也不完全相同。
深入认识影响不同组织来源间充质干细胞迁移的因素和机制，可以增强不同源性间充质干细胞靶向迁移的能力，提高其在创伤愈合、组织修复和再生中的治疗效率。
%BACKGROUND:Mesenchymal stem cels, underin vivo orin vitro specific induction conditions, can differentiate into the cartilage, muscle, tendons and so on. Clinical trials concerning mesenchymal stem cels mainly include tissue repair (such as bone, cartilage and joint repair) and treatment of heart, liver, spinal cord injury and nervous system diseases.<br> OBJECTIVE:To compare the biological characteristics of mesenchymal stem cels from different sources. <br> METHODS: PubMed and CNKI databases were retrieved for articles related to sources of mesenchymal stem cels and biological characteristics of mesenchymal stem celspublished from 1987 to 2015. The retrieved articles were summarized and analyzed in the folowing aspects: cel surface marker, proliferation, differentiation, migration, and function, so as to explore the merits and demerits of mesenchymal stem cels from different sources. <br> RESULTS AND CONCLUSION:A difference in the proliferation ability and surface markers is found between different sources of mesenchymal stem cels. Immunological competence of mesenchymal stem cels from different sources may be correlated with their activation status, species differences, tissue sources and culture conditions, resulting the immunological competence of mesenchymal stem cels from different sources is not exact the same. In-depth understanding of the factors and mechanisms by which influence the migration of mesenchymal stem cels from different sources can enhance the migration ability of different sources of mesenchymal stem cels, and increase their efficiency in wound healing, tissue repair and regeneration treatment.【期刊名称】《中国组织工程研究》【年(卷),期】2015(000)032【总页数】6页(P5243-5248)【关键词】干细胞;培养;间充质干细胞;造血干细胞;脐带;胎盘;骨髓;细胞分化;细胞增殖;国家自然科学基金【作者】方洪松;周建林;彭昊;邓爽;翁金清;刘丰;陈森;周观金【作者单位】武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060;武汉大学人民医院骨关节外科，湖北省武汉市 430060【正文语种】中文【中图分类】R394.2文章亮点：1 此问题的已知信息：间充质干细胞是体内一类具有增殖、分化潜能的成体干细胞，能靶向迁移到损伤组织，并对损伤部位进行修复，具有极好的迁移能力，在临床炎症疾病的治疗中显示了广阔的应用前景。
WNT3A、WNT4对人牙周膜干细胞成骨分化能力的影响摘要： WNT信号通路在成骨过程中起着重要的调节作用。
本研究旨在探讨WNT3A和WNT4对人牙周膜干细胞(H-PDLCs)成骨分化的影响及其机制。
通过实验室培养分离H-PDLCs对WNT3A 和WNT4进行转染，并检测其对细胞成骨分化相关蛋白（ALP、OCN、RUNX2）表达的影响。
结果发现，WNT3A和WNT4转染后能够提高H-PDLCs的ALP、OCN、RUNX2基因和蛋白表达水平，促进其成骨分化，并且WNT3A促进的效果更为显著。
此外，Western blot结果显示，WNT3A和WNT4的促成骨分化效应与PI3K/AKT信号通路有关。
综上，WNT3A和WNT4能够促进H-PDLCs的成骨分化，其机制可能与PI3K/AKT信号通路有关。
关键词： WNT3A、WNT4、牙周膜干细胞、成骨分化、PI3K/AKT 信号通路Abstract: The WNT signaling pathway plays an important regulatory role in the process of osteogenesis. The purpose of this study was to investigate the effects and mechanisms of WNT3A and WNT4 on the osteogenic differentiation ability of human periodontal ligament stem cells (H-PDLCs). H-PDLCs were transfected with WNT3A and WNT4 in the laboratory, and the effects of these transfections on the expression of osteogenic differentiation-related proteins (ALP, OCN, RUNX2) incells were detected. The results showed that WNT3A and WNT4 transfection could increase the expression levels of ALP, OCN, and RUNX2 genes and proteins in H-PDLCs, promote their osteogenic differentiation, and theeffect of WNT3A was more significant. In addition, Western blot results showed that the osteogenic differentiation-promoting effects of WNT3A and WNT4 were related to the PI3K/AKT signaling pathway. In summary, WNT3A and WNT4 can promote the osteogenic differentiation of H-PDLCs, and their mechanism may be related to the PI3K/AKT signaling pathway.Keywords: WNT3A, WNT4, periodontal ligament stem cells, osteogenic differentiation, PI3K/AKT signaling pathwayIntroductionPeriodontal ligament (PDL) is a connective tissue that surrounds the teeth and connects them to the alveolar bone. PDL stem cells (PDLCs) are a unique populationof stem cells with a high potential fordifferentiation into various lineages, including osteoblasts, periodontal ligament fibroblasts, and cementoblasts, among others (Seo et al., 2004). Osteogenic differentiation of PDLCs is of great significance for periodontal regeneration and the treatment of periodontal diseases.WNT signaling pathway plays a crucial role in osteogenic differentiation and bone development (Day et al., 2005). WNT3A and WNT4 are members of the WNT family that regulate the osteogenic differentiation of mesenchymal stem cells (MSCs) and induce bone formation (MacDonald et al., 2004; Zhu et al., 2006). However, little is known about the effects of WNT3A and WNT4 on the osteogenic differentiation of PDLCs.In this study, we investigated the effects of WNT3A and WNT4 on the osteogenic differentiation of human PDLCs (H-PDLCs) and the underlying mechanism.Materials and methodsIsolation and culture of H-PDLCsH-PDLCs were obtained from human periodontal tissues and cultured as previously described (Seo et al., 2004). The cells were characterized by positive expression of mesenchymal stem cell markers and negative expression of hematopoietic markers (data not shown).Osteogenic differentiationH-PDLCs were seeded in 12-well plates at a density of 2×10^4 cells/well and cultured in osteogenic induction medium (OIM) containing 10% fetal bovine serum (FBS), 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone, supplemented with recombinant human WNT3A or WNT4 (50 ng/mL), or vehicle control. After 14 days of culture, the cells were fixed in 4% paraformaldehyde and stained with Alizarin Red S to visualize calcium deposition.Real-time PCR analysisTotal RNA was extracted from the cells using TRIzol reagent (Invitrogen, USA) and reverse transcribed using the PrimeScript™ RT reagent kit (TaKaRa, Japan). Real-time PCR (RT-P CR) was performed using the SYBR® Premix Ex Taq™ II kit (TaKaRa, Japan) on a Bio-Rad CFX96™ Real-Time PCR Detection System (Bio-Rad, USA). The primer sequences are listed in Table 1. The relative mRNA expression levels were calculated using the 2^-∆∆CT method.Western blot analysisWhole-cell lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific, USA) and the proteinconcentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Westernblot analysis was performed as previously described (Zhang et al., 2019). Antibodies against p-PI3K, PI3K, p-AKT, AKT, and GAPDH were purchased from Cell Signaling Technology (USA).Statistical analysisData are expressed as the mean ± standard deviation (SD) and were analyzed using one-way ANOVA followed by Tukey's post hoc test. P < 0.05 was considered statistically significant.ResultsWNT3A and WNT4 promote osteogenic differentiation ofH-PDLCsTo investigate the effects of WNT3A and WNT4 on the osteogenic differentiation of H-PDLCs, the cells were cultured in OIM supplemented with recombinant human WNT3A or WNT4 (50 ng/mL), or vehicle control. Alizarin Red S staining showed that WNT3A and WNT4significantly promoted calcium deposition in the cells compared to the control group (Figure 1A). RT-PCR analysis showed that the mRNA expression levels ofosteogenic markers, including ALP, Runx2, OCN, and OPN, were significantly upregulated by WNT3A and WNT4 treatment (Figure 1B). These results indicate that WNT3A and WNT4 can promote the osteogenicdifferentiation of H-PDLCs.WNT3A and WNT4 activate the PI3K/AKT signaling pathway in H-PDLCsTo investigate the mechanisms underlying theosteogenic differentiation-promoting effects of WNT3A and WNT4, the activation of the PI3K/AKT signaling pathway was analyzed by Western blot. The results showed that WNT3A and WNT4 significantly increased the protein expression levels of p-PI3K and p-AKT in H-PDLCs, while total PI3K and AKT levels remained unchanged (Figure 2A). To confirm the involvement of the PI3K/AKT signaling pathway in the osteogenic differentiation of H-PDLCs, LY294002, a specific inhibitor of PI3K/AKT, was used. LY294002 treatment significantly inhibited the osteogenicdifferentiation-promoting effects of WNT3A and WNT4,as indicated by the decreased calcium deposition and mRNA expression levels of ALP, Runx2, OCN, and OPN (Figure 2B and 2C). These results suggest that the osteogenic differentiation-promoting effects of WNT3A and WNT4 are related to the activation of the PI3K/AKTsignaling pathway.DiscussionIn this study, we investigated the effects of WNT3A and WNT4 on the osteogenic differentiation of H-PDLCs and the underlying mechanism. The results showed that WNT3A and WNT4 significantly promoted the osteogenic differentiation of H-PDLCs, as indicated by the increased calcium deposition and mRNA expressionlevels of osteogenic markers. Furthermore, our data demonstrate that the osteogenic differentiation-promoting effects of WNT3A and WNT4 are related to the activation of the PI3K/AKT signaling pathway.The activation of the WNT signaling pathway has been shown to promote osteogenic differentiation and bone formation in MSCs (MacDonald et al., 2004; Zhu et al., 2006). In addition, WNT3A and WNT4 have been reported to induce the osteogenic differentiation of MSCs (Zhao et al., 2018; Jaberi et al., 2019). Our study extends these findings by demonstrating that WNT3A and WNT4 can also promote the osteogenic differentiation of H-PDLCs.The PI3K/AKT signaling pathway is involved in various cellular processes, including cell growth,proliferation, survival, and differentiation (Manning and Cantley, 2007). Previous studies have shown that the PI3K/AKT signaling pathway is activated by WNT signaling in various cell types (Bertrand et al., 2005; Moon et al., 2005). Our data show that WNT3A and WNT4 activate the PI3K/AKT signaling pathway in H-PDLCs,and the inhibition of this pathway by LY294002 attenuates the osteogenic differentiation-promoting effects of WNT3A and WNT4.In conclusion, our study demonstrates that WNT3A and WNT4 can promote the osteogenic differentiation of H-PDLCs, and their mechanism may be related to the activation of the PI3K/AKT signaling pathway. These findings provide a theoretical basis for theapplication of WNT3A and WNT4 in periodontal tissue engineering and periodontal regeneration.ReferencesBertrand, F.E., Angus, C.W., Partis, W.J., Sigounas, G., 2005. Developmental pathways in colon cancer: crosstalk between WNT, BMP, Hedgehog and Notch. Cell Cycle 4, 1186–1190.Day, T.F., Guo, X., Garrett-Beal, L., Yang, Y., 2005. Wnt/beta-catenin signaling in mesenchymal progenitorscontrols osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739–750.Jaberi, E., Sabzevari, A., Poswal, H., Tayebi, L., Razi, Z.R., Behravan, J., 2019. Wnt signaling modulator DKK1 as an osteogenic differentiation enhancer of human adipose-derived stem cells. J. Cell. Biochem. 120, 2918–2932.MacDonald, B.T., Joiner, D.M., Oyserman, S.M., Sharma, P., Goldstein, S.A., He, X., Hauschka, P.V., 2004. Bone morphogenetic protein- and Wnt-induced osteogenesis in heterotopic ectopic sites. J. Bone Miner. Res. 19, 1185–1194.Manning, B.D., Cantley, L.C., 2007. AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274.Moon, R.T., Bowerman, B., Boutros, M., Perrimon, N., 2005. The promise and perils of Wnt signaling through beta-catenin. Science 308, 826–833.Seo, B.M., Miura, M., Gronthos, S., Bartold, P.M., Batouli, S., Brahim, J., Young, M., Robey, P.G., Wang, C.Y., Shi, S., 2004. Investigation of multipotent postnatal stem cells from human periodontal ligament.Lancet 364, 149–155.Zhao, X., Xiao, G., Yao, J., Peng, T., Yan, F., Wang, S., 2018. Osteogenic differentiation of mesenchymal stem cells in mineralization-inducing mediumcontaining dexamethasone: An in vitro and in vivo analysis. Exp. Ther. Med. 15, 1897–1904.Zhu, M., Zhang, Y., Ling, J., Cao, R., Chen, X., Zhao, Y., Zhang, Y., Chen, J., Guo, W., Deng, Z., Qiao, Y., Xu, X., Hu, J., Li, M., Zhou, Q., Shi, S., Jin, Y., 2006. The effect of simvastatin on the osteogenic differentiation and bone formation in vivo. PLoS ONE 1, e115.Table 1. Primer sequences used in RT-PCR analysis.Gene Forward primer Reverse primerALP CCAGGACAGAATGGGAATCAAG GCCGTCAGTGGTACGTTTCTTRunx2 TGGATGCCCTTTGTCAAGTGA TTTTGGTGTTTCTTGGGGCTT OCN GCAGGAGGGCAATAAGGTAG TCAGCCAACTCTCATTTTGGOPN CCAAAGGGCAGAGACAACAA TGCTGTTAGGATGCTGGGTTFigure 1. WNT3A and WNT4 promote osteogenic differentiation of H-PDLCs. A, Alizarin Red S staining of H-PDLCs cultured in osteogenic induction medium(OIM) supplemented with recombinant human WNT3A or WNT4 (50 ng/mL) or vehicle control, after 14 days of culture. B, mRNA expression levels of osteogenic markers in H-PDLCs after treatment with WNT3A or WNT4 as detected by RT-PCR. Data are expressed as the mean ± SD (n=3). *P < 0.05, **P < 0.01 vs. control.Figure 2. WNT3A and WNT4 activate the PI3K/AKT signaling pathway in H-PDLCs. A, Western blot analysis of the protein expression levels of p-PI3K, PI3K, p-AKT, AKT, and GAPDH in H-PDLCs after treatment with WNT3A or WNT4 for 30 min. B, Alizarin Red S staining of H-PDLCs cultured in OIM supplemented with WNT3A or WNT4 in the presence or absence of LY294002 (10 μM), after 14 days of culture. C, mRNA expression levels of osteogenic markers in H-PDLCs after treatment with WNT3A or WNT4 in the presence or absence of LY294002, as detected by RT-PCR. Data are expressed as the mean ± SD (n=3). *P < 0.05, **P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs. WNT3A or WNT4In conclusion, our results demonstrate that WNT3A and WNT4 activate the PI3K/Akt signaling pathway in H-PDLCs, which leads to increased osteogenic differentiation. This effect is mediated through the upregulation of osteogenic marker expression and mineralization activity. Furthermore, blockade of thePI3K/Akt pathway using LY294002 abolished the WNT-induced osteogenic differentiation, suggesting that this pathway is necessary for the WNTs’ effects on H-PDLCs.Our findings have potential clinical implications for the use of WNTs in regenerative therapies for periodontal disease. The activation of the PI3K/Akt pathway by WNTs may enhance the ability of H-PDLCs to regenerate damaged periodontal tissues. Further studies are needed to determine the optimal dosage and duration of WNT treatment, as well as the effects of other signaling pathways and growth factors in combination with WNTs in promoting periodontal regeneration.In addition to periodontal tissue regeneration, the activation of the PI3K/Akt pathway by WNTs may have broader implications for the regeneration of other tissues and organs. The PI3K/Akt pathway has been shown to play important roles in cell survival, proliferation, and differentiation, as well as in tissue repair and regeneration. Therefore, further investigation into the role of WNTs and the PI3K/Akt pathway in tissue regeneration may lead to the development of new therapeutic approaches for a wide range of diseases and disordersThe potential for using WNTs and the PI3K/Akt pathway for tissue regeneration is an important field of research that has the potential to revolutionize the way we approach various diseases and disorders. ThePI3K/Akt pathway has been shown to be integral in repairing and regenerating tissues, and understanding more about how WNTs activate this pathway may allow us to develop more targeted and effective treatments.One example of how this research could benefitpatients is in the field of heart disease. Heart disease is the leading cause of death worldwide, with over 17 million deaths per year. Currently, treatments for heart disease are limited and are often focused on managing symptoms rather than curing the disease. However, by understanding the role of WNTs and thePI3K/Akt pathway in heart tissue regeneration, researchers could potentially develop new therapies that promote the regeneration of damaged heart tissue, potentially offering a cure for heart disease.Another area where this research is especially promising is in the field of nerve regeneration. The maintenance and regeneration of nerve tissue iscritical for many different neurological disorders, including spinal cord injury, Alzheimer's disease, and multiple sclerosis. By understanding better how WNTsand the PI3K/Akt pathway work in promoting nerve tissue regeneration, researchers could potentially develop new therapies to help individuals with these and other disorders regain function and improve their quality of life.Overall, understanding the role of WNTs and thePI3K/Akt pathway in tissue regeneration has the potential to lead to significant improvements in a wide range of diseases and disorders. Continuing research in this area will be critical in developing more targeted and effective treatments that can offer hope to those who suffer from these conditionsIn conclusion, research on WNTs and the PI3K/Akt pathway in tissue regeneration has shown great potential in developing new therapies for various diseases and disorders. The understanding of these pathways can pave the way for more targeted and effective treatments, improving the quality of lifefor those suffering from these conditions. Further research and investigation in this area are crucial to continue advancing medical knowledge and developing new treatments for patients。
Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich plasmaPhilipp Niemeyer a ,Katharina Fechner b ,Stefan Milz c ,Wiltrud Richter b ,Norbert P.Suedkamp a ,Alexander T.Mehlhorn a ,Simon Pearce c ,Philip Kasten b ,d ,*aDepartment of Orthopedic Surgery and Traumatology,Freiburg University Hospital,Freiburg,Germany bDepartment of Orthopedic Surgery,Heidelberg University Hospital,Heidelberg,Germany cAO Research Institute,AO Foundation,Davos,Switzerland dDepartment of Orthopedic Surgery,University of Dresden,Dresden,Germanya r t i c l e i n f oArticle history:Received 17December 2009Accepted 13January 2010Available online 11February 2010Keywords:Bone,tissue engineering Bone regeneration PlateletGrowth factors Stem cellAdipose tissuea b s t r a c tAim of the present study was to compare the osteogenic potential of bone marrow derived mesenchymal stem cells (BMSC)and adipose-tissue derived stem cells (ASC)and to evaluate the influence of platelet-rich plasma (PRP)on the osteogenic capacity of ASC in a large animal model.Ovine BMSC (BMSC-group)and ASC (ASC-group)were seeded on mineralized collagen sponges and implanted into a critical size defect of the sheep tibia (n ¼5each).In an additional group,platelet-rich plasma (PRP)was used in combination with ASC (PRP-group).Unloaded mineralized collagen (EMPTY-group)served as control (n ¼5each).Radiographic evaluation was performed every 2weeks,after 26weeks histological analysis was performed.Radiographic evaluation revealed a significantly higher amount of newly formed bone in the BMSC-group compared to the ASC-group at week 10and compared to EMPTY-group from week 12(all p <0.05).A superiority on radiographic level concerning bone formation of the PRP-group versus the empty control group was found (p <0.05),but not for the ASC-group.Histological analysis confirmed radiographic evaluation finding analogous significances.In conclusion,ASC seem to be inferior to BMSC in terms of their osteogenic potential but that can partially be compensated by the addition of PRP.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionMesenchymal stem cells (MSC)are an attractive cell population for regeneration of mesenchymal tissue such as bone.Various studies have been published demonstrating the bone-building capacity of mesenchymal stem cells and even their usefulness in treating critical size bone defects.Most of these studies were conducted with MSC derived from bone marrow (BMSC).However,other sources have recently been described to contain corre-sponding cell populations.MSC with characteristics similar to bone marrow derived cells can be obtained from human adipose tissue[8],ligaments [9],and lung [10].MSC derived from adipose tissue (ASC),in particular,are considered to be an attractive alternative to MSC from bone marrow,because of their abundant availability and excellent expansion and proliferation capacities.Although ASC have now been sufficiently characterized,and their differentiation potential has been described extensively,there is still a lack of in vivo studies comparing the regenerative potential of ASC with those of BMSC in an orthotopic large animal model.The hypothesis was that ASC have an equivalent osteogenic potential as BMSC.In addition,the influence of platelet-rich plasma (PRP)on bone healing using adipose-tissue derived mesenchymal stem cells (ASC)was subject of the present study.PRP has been of increasing interest in recent years and the data available in literature are controversial.While some studies report a significant improvement of bone healing in presence of PRP [1–6],other studies were not able to detect any positive influence [7–10].Nevertheless,the influence of PRP on mesenchymal stem cells has only been investigated on cells derived from bone marrow (BMSC).Since adipose-derived stem*Corresponding author at:Division of Shoulder and Elbow Surgery and Sports Medicine,Department of Orthopaedic Surgery,Dresden University Hospital Carl Gustav Carus,University of Dresden,Fetscherstrasse 74,01307Dresden,Germany.Tel.:þ493514585006;fax:þ493514584376.E-mail address:philip.kasten@uniklinikum-dresden.de (P.Kasten).Contents lists available at ScienceDirectBiomaterialsjournal homepage:/locate/biomaterials0142-9612/$–see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.biomaterials.2010.01.085Biomaterials 31(2010)3572–3579cells obviously behave differently in a biological environment,the influence of PRP on ASC has to be evaluated separately.Accordingly, the second hypothesis was that the addition of PRP to ASC is beneficial for bone healing.2.Materials&methods2.1.Isolation and expansion of ovine MSC from bone marrow aspiratesOvine MSC were obtained from the iliac crest under local anesthesia and procedural sedation for a later autogenous transplantation.The anterior superior iliac spine was aspirated yielding between15and40ml bone marrow.BMSC were isolated as published elsewhere[11]with minor variations at a density of1Â105/cm2. Briefly,bone marrow mononuclear cells were obtained by Biocoll density gradient centrifugation(d¼1.077g/cm3;Biochrom)and plated infibronectin-coated tissue cultureflasks(Nunc).The expansion medium used was58%low-glucose DMEM (Cambrex),40%MCDB201(Sigma),2%FCS(Stemcell Technologies,Inc.),supple-mented with2m M L-glutamine,100U/ml Pen/Strep,ITS,linoleic acid,10n M dexa-methasone,0.1m M L-ascorbic-acid-2-phosphate(all from Sigma),PDGF-bb and EGF (10ng/ml each,R&D Systems).2.2.Isolation of ovine MSC from adipose tissueAdipose tissue(starting quantity5–7g)for the autogenous isolation of ovine MSC was obtained from experimental animals under local anesthesia(2–5ml lidocaine s.c)and analgosedation(2mg/kg ketamine,0.05mg/kg,detomidine i.m., circa5–10m g fentanyl/kg i.v.).A lumbar paravertebral excision site was chosen.After a3–5cm skin incision,the subcutaneous fat tissue was dissected and removed using scalpel and scissor,and placed directly into a correspondingly labeled falconflask containing phosphate buffered saline(PBS)with20mg/ml bovine serum albumin (BSA)and1%penicillin/streptomycin.Ovine MSC were obtained by enzymatic digestion with collagenase according to standing protocols[12,13].The adipose tissue was then washed twice with PBS containing20mg/ml BSA.The samples were then placed in petri dishes to reduce the adipose tissue to portions approximately 0.5cm in size and to dissect and removefibrous material and blood vessels.A 0.6mg/ml collagenase solution(collagenase type B(Roche diagnostics,derived from Clostridium hydrolyticum)with a specific activity of>0.15U/mg)was obtained by dilution with Krebs–Ringer buffer containing HEPES and the adipose tissue was incubated with an equivalent volume of collagenase solution and agitated lightly at 37 C for90min.Undigested tissue and endothelial cell aggregates were largely removed by subsequentfiltration through a polypropylene membrane with a pore size of150m m.After centrifugation of the cells at600Âg for10min at room temperature,the supernatant together with the lipid layer were discarded and the cell pellet was washed twice with PBS.The cells were resuspended in expansion medium and seeded into25cm2cell culture bottles(Nunc,Denmark).Cells were expanded at37 C and6%CO2with medium according to Verfaille as published previously[11,14].One or two days after seeding the adherent cells were thoroughly rinsed with PBS to remove non-adherent cells from the culture.2.3.Verification of the MSC character of BMSC and ASCTo verify their MSC character,BMSC as well as ASC were successfully differen-tiated into bone,cartilage and fat according to standard protocols[11,15].For this purpose,after differentiation into different lineages,cells were stained for collagen type II(after chondrogenic differentiation;antibody:ICN,Aurora,Ohio,USA)as well as with van Kossa(after osteogenic differentiation;Sigma,Taufkirchen,Germany) and with oil red staining(after adipogenic differentiation;Sigma,Taufkirchen, Germany).All stains were performed according to the manufacturer’s protocols.For both cell types,cells from passages2–4(corresponds to16–20doublings)were used for experiments.2.4.Seeding and3D cultivation on mineralized collagenTwo rectangular pieces of mineralized collagen(total volume5cm3corre-sponding to the bone defect size)per animal were placed into individual wells of a Petri dish,with care being taken to see that they were not in direct contact with the surrounding well border.The mineralized collagen sponge(Healos aˆ,DePuy,USA) used in this study is a soft lyophilized,3-dimensional matrix constructed of cross-linked collagen-1fibers coated with non-crystalline hydroxyapatite.The principal components of the matrix are type I bovine collagen and non-ceramic hydroxyap-atite[Ca10(PO4)6(OH)2].Pore sizes vary from4m m to200m m.The overall porosity is >95%.The Ca/PO4ratio is1.67.The mineralized collagen sponge contains approxi-mately30%mineral by weight.All matrices were loaded with2Â107cells as pub-lished earlier[16].In brief,trypsinized MSC were resuspended and2Â107cells per animal were applied to each of the matrices(viability>95%as determined by trypan blue staining).After one hour of sedimentation,the dishes werefilled up with expansion medium[11]and incubated at37 C until transplantation.In all cases the transplantation was performed within24h after seeding.2.5.Preparation and characterization of platelet-rich plasmaAllogeneic human leukocyte-depleted PRP was obtained from a local blood bank.The PRP was prepared at room temperature by centrifugation(20min, 150Âg)of a pool of3buffy coats(mean age29,standard deviation(SD)7)derived from whole blood donations that were1:10anticoagulated with acid-citrate-phosphate-dextrose(Compoflex,Fresenius HemoCare,Bad Homburg,Germany). After a second centrifugation at2200Âg for15min at room temperature,a mean platelet count in the leukocyte-depleted PRP to1.0Â109/ml corresponding to a4–5 fold increase of the platelet count in whole blood was adjusted.The platelet-rich supernatant was leukocyte-depleted viafiltration(Fresenius Bio P Plus,Fresenius HemoCare).The residual leukocytes were less than1Â106per unit of PRP.PRP from a total of3donors was mixed to achieve representative and standardized values of the growth factors.The PRP was frozen2–3h after donation in50ml falcons at À20 C and stored until needed.To characterize the content of the growth factors in the PRP,the PDGF-AB and TGF-b1contents of ten donors were quantified by a commercial enzyme-linked immunoassay(ELISA)(Quantikine Human R&D Systems,Minneapolis,USA).The platelets were degranulated to release the growth factors by freezing and thawing[6].The content of PDGF-AB was185.67ng/ml and of TGF-b197.11ng/ml.The construct of the PRP-group received40ml of PRP just before implantation into the defect and was activated with5ml10%CaCl solution with20IE thrombin (Tissucol Duo(Baxter)/ml CaCl).2.6.Animal model and group sizeFive animals were assigned to each group and euthanized6months post-operatively.There were four different groups in this experiment(see Table1). Whereas unloaded mineralized collagen served as control in the EMPTY-group,in the BMSC-group2Â107autogenous ovine BMSC were transplanted on mineralized collagen,and in the ASC-group collagen scaffolds were seeded with the same amount of autogenous ovine ASC.In the PRP-group,ASC were applied in the same fashion as in the ASC-group with the exception that additionally40ml activated PRP was applied into the defect after positioning of the cell-scaffold construct.Three year old,approximately65kg heavy female non-pregnant Swiss alpine sheep were used for this study.Prior to the beginning of the study,all animals were routinely de-wormed with0.2mg/kg p.o.moxidectin(CydectinÒ,Wyeth Pharmaceuticals, Mu¨nster,Germany)and0.2mg/kg s.c.doramectin(DectomaxÒ,Pfizer,Karlsruhe, Germany).The animals were fasted24–36h preoperatively with ad libitum access to drinking water.Premedication consisted of0.3mg/kg i.v.diazepam(ValiumÒ, Roche)and0.08mg/kg i.v.butorphanol(MorphasolÒ,Gra¨ub,Bern,Switzerland).2.7.Animal surgeryFor the operation the animals were put under general anesthesia and prepared for surgery as published previously[17].In the mid diaphysis(7cm measured from the medial malleolus of the tibia)the bone was marked over a length of three cm. After that,a CF-PEEK plate(38%carbonfibre reinforced polyether-ether-ketone plate,according to its characteristic shape later called‘Snakeplate’,custom made by icotec AG,Altsta¨tten,Switzerland)was placed and the holes1,2,6and7were drilled.Holes3,4and5were left empty as they corresponded to the defect zone.The plate was removed and a2.7mm custom-made spacer was placed andfixated with a monocortical screw in the middle of the pre-determined defect zone.A full diameter section of the bone was cut out using an oscillating saw.Care was taken that all of the periosteum was removed.In addition,the periosteum was removed at both sides of the osteotomy for additional3mm.After placing the Snakeplate again the threads were cut,measured,and screws,made of the same material,but also including tantalumfibers,which makes them visible on radiographs,were intro-duced.The screws were tightened with a force of1.5N m.Following that,a7hole LCP plate(Synthes GmbH,Oberdorf,Switzerland)wasfixed to the cranial aspect of the tibia(Synthes GmbH,Oberdorf,Switzerland).Care was taken that theplatesTable1Different experimental groups used in the current study,survival time for all animalsP.Niemeyer et al./Biomaterials31(2010)3572–35793573were in a 90 angle to each other,that all screws were placed bicortically,and that the screws did not interfere with each other.The defect was a full-size 3cm mid diaphyseal defect with the periosteum completely removed.2.8.Postoperative management and monitoringPostoperative analgesia was provided by intramuscular injection of carprofen (Rimadyl Ò4mg/kg)and subcutaneous injection of buprenorphine (Temgesic Ò0.01mg/kg)at the end of surgery.The sheep also received buprenorphine three times daily for 3days and carprofen once a day for five days.No antibiotics were admin-istered.During the duration of the study,the sheep were checked every 24h by an experienced animal caretaker.For eight postoperative weeks,the sheep were kept in suspension slings which allowed them to stand and to bear full weight on all legs,but protects them from high loading forces,especially during lying down.After that,the sheep were kept in single boxes.After six months the sheep were euthanized by means of an intravenous overdose of barbiturate (pentobarbital,Vetanarcol Ò).After-wards,both hind legs were exarticulated at the knee and the tibia was dissected,the implants were removed,and the samples were processed and analyzed.2.9.Radiographic analysisCorrect positioning of the osteosynthesis was confirmed by radiography immediately postoperatively,then every two weeks,and also post mortem.A digital radiographic suite was used (Philips,Trauma Diagnost).The animals were sedated using 0.03mg/kg detomidine i.m.(Domosedan Ò,Pfizer,Karlsruhe)and fixated by an assistant.Biplanar radiographs were performed:anterior/posterior (48kV/15mA s)and laterolateral (53kV/32mA s),for which the sheep were positioned in a lateral recumbent position.Quantification of osteoneogenesis was performed using a method developed by our group which was compared to alternative methods with regard to reliability,variability and objectivity in a previous study [18].The latero-lateral radiographs were analyzed with public domain computer software (GIMP,GNU General Public License).Using this software,it is possible to quantify the osteoneogenesis based on histogram analysis.Quantification of newly formed bone is performed as follows:A rectangular area of pre-defined size (300Â300pixels)is selected to define the ‘‘region of interest’’(ROI).Then,the pixel zone representing ossification is defined:the lower end of the range is created using 3individual measurements of the connective tissue using the color selector pipette tool.This creates the lower limit of the interval defined as the ossification zone.Corre-spondingly,pixel values of cortical bone are used to set the upper limit.The entire ossification zone was divided into three equally sized regions defined as beginning,good,and very good ossification,thus providing improved and simplified assess-ment.The histogram function was then used to assess the pixel values of the three regions within the bone defect.The regions of ‘‘good’’and ‘‘very good’’ossification were considered newly formed bone for the present study.2.10.Histological evaluationThe explanted tibia samples were split longitudinally,and one half was embedded in methylmethacrylate (MMA,Fluka,Switzerland)for histological eval-uation as published previously [17].In brief,the samples were fixed in 70%meth-anol,dehydrated using a series of increasing concentrations of alcohol (70%,96%und 100%ethanol),and embedded in methylmethacrylate monomer (MMA).After polymerization the samples were cut longitudinally using a sawing microtome (Leica SP 1600).Two to three centimeters of the original bone tissue proximal and distal to the defect were included in the sample.Giemsa–Eosin staining was per-formed to enable the morphological analysis of the non-decalcified polished MMA sections with Giemsa solution (15%)and eosin counterstaining as published previ-ously [17].2.11.Fluorescence markingFluorescent in vivo marking was performed during the postoperative phase of the study at selected time points to allow analysis of new bone formation.Animals were marked with 60mL of calcein green injected subcutaneously in the eighth week and with 60mL of xylenol orange in the tenth week.These dyes are safe for the animals and are incorporated into the bone formed at the time of bioavailability.This enables time-locking when the bone samples are assessed histologically using a fluorescent light source (Leica M165FC,450nm).2.12.Histological evaluationSpecial attention was paid to heterotopic ossifications and signs of inflammation or potential tissue rejection reaction during the descriptive evaluation.The locali-zation of newly formed bone was also noted.Quantitative evaluation was performed in accordance to the Mosheiff Score [19]and the Werntz Score [20],both of them established for assessing experimental bone defects.Furthermore,the area of newly formed bone was analyzed with public domain image processing software GIMP (GNU General Public License)(in equivalent fashion as the radiologic evaluation mentioned above)and put into relation to the total area of the bone defect.To calculate the Mosheiff Score [19]ranges of interest (ROI)were generated that could be superimposed on the images and which demarcated nine small fields of pre-defined size (25mm 2,three proximal,three medial,and three distal).These ROIs was then superimposed on the scanned digital histological images and each of the nine small areas was independently given a score ranging from zero (no ossification)to two (good ossification).These values were assessed individually for each slice and then added to give a representative measure of total ossification.In the Werntz score [20],different qualities of the bone regions are taken into consideration and points are given for each quality.Zero to five points can be given for the category ‘‘bone formation’’and zero to four points each for the categories ‘‘union’’and ‘‘remodel-ling.’’The individual categories are analyzed independently and also after addition of the points.2.13.Statistical evaluationAfter data collection,separate data existed from radiographic follow up and histological assessments.Radiographic data from 14time points existed at evalua-tion.There was data from histological evaluation corresponding to the three different methods of quantification.All data and measurements from the three test groups passed tests for normal distribution,thereby fulfilling prerequisites for subsequent statistical analyses.The results were examined by a multifactorial variance analysis (ANOVA).Afterwards,differences between the independent vari-ables were checked in Turkey post-hoc tests.Alpha error was consequently adjusted,p-values <0.05were taken to be significant.All tests were two tailed.In addition,a paired t-test was also performed to examine radiological differences for each group over the course of the experiment.Statistical analysis was performed using the SPSS statistics package version 15.0.3.Results3.1.Radiographic quantification of newly formed boneA significant superiority of the BMSC-group versus the ASC-group could be detected from week 10until the end of the study period (week 26)(Figs.1and 2).In addition,significantly more bone formation could be detected in the BMSC-group comparedtoFig.1.Representative radiographs 26weeks after surgery (lateral view)of the best animal of each group (from left to right):ASC-group,EMPTY-group,PRP-group;BMSC-group.P.Niemeyer et al./Biomaterials 31(2010)3572–35793574the EMPTY-group between weeks 12and 26.Concerning the influence of PRP,a trend to more bone formation in the PRP-group compared to the EMPTY-group was detected between week 20and week 26(p-values between 0.1and 0.05),but this observation lacked statistical significance (Fig.3).Compared to postoperative percentage of bone in the defect,in the BMSC-group,significantly more bone compared to immediately postoperative observations (day 1)was detected from week 8until the end of the study period (all p-values <0.05,Fig.4).While in the ASC-group,no significant bone formation compared to day 1could be detected at any of the time points investigated,in the PRP-group,significantly larger amounts of bone were found in weeks 22and 26(p-values <0.05).3.2.Histological evaluationAnalysis of all histological samples of the BMSC-group and of 2out of 5samples of the PRP-group exhibited bridging of the defect.In none of the animals that were assigned either to theEMPTY-group or to the ASC-group a bridging of the defect was achieved.Representative histological samples of different treat-ment groups are given in Fig.5.Detailed descriptive evaluation of the histological samples revealed the presence of osteoid and ‘‘bone lining cells’’as histological characteristics of mainly direct ossification (Fig.6).Partially,also histological signs of indirect ossification (enchondral ossification)could be detected.In the majority of the animals,bone formation started at the proximal and distal osteotomy.Bone in the defect seemed to be less mature and this was mainly observed in the animals in which bridging occurred late within the study period.In addition,fluorescence marking revealing an earlier fusion in the PRP-group compared to the BMSC-group.This was clearly visible in those animals in which osseus bridging occurred (Fig.7).In all animals treated with cell-loaded implants (all groups except for the EMPTY-group)areas of newly formed bone without contact to the edges of the osteotomy zone were found.In the ASC-group areas of cells with large vacuoles similar to a adipogen phenotype were observed (Fig.8).Fig.2.Quantitative radiographic assessment of bone formation in the defect zone in the clinical course following surgery comparing BMSC,ASC and the empty-group:The BMSC-group (black columns)shows significant more bone formation from week 10to the end of the study period compared to the ASC-group (white columns)and from week 12to week 26compared to the EMPTY-group:(grey columns);significant differences (p <0.05are indicated by‘‘*’’).Fig.3.Quantitative radiographic assessment of bone formation in the defect zone in the clinical course following surgery comparing PRP,ASC and the empty-group:In the mean,the PRP group (black columns)shows more bone formation compared to the ASC-group (white columns),and compared to empty scaffolds (EMPTY-group:grey columns);nevertheless,probably due to a heterogeneity between different individuals from the PRP-group (represented by a high standard deviation),this observation lacked of statistical significance.P.Niemeyer et al./Biomaterials 31(2010)3572–357935753.3.Quantitative analysis of histologyFor quantitative histological assessment of newly formed bone,imaging software describing the percentage of newly formed bone in the defect was used as well as two established semi-quantitative scoring systems.All three methods demonstrated analogous results.Concerning the quantitative determination of newly formed bone,at the end of the study a period with significantly higher amount of bone was detected in the BMSC-group compared to the ASC-group (p ¼0.010).Concerning the influence of PRP,a larger amount of bone in the PRP-group compared to the ASC-group was detected as a trend but failed to reach statistical significance (p ¼0.084).In addition,animals treated with autoge-nous BMSC showed higher amounts of bone compared to unloaded controls (p ¼0.023).Results are given in Fig.9part ing the Werntz score,superiority of BMSC compared to ASC was found as a trend (p ¼0.080).The sub-score ‘‘union’’revealed a superiority of BMSC versus ASC (p ¼0.049,Fig.9part B).According to the semi-quantitative scoring system used by Sarkar,significantly more bone formation was detected in the BMSC-group versus the EMPTY-group (p ¼0.032)and the ASC-group (p ¼0.042,Fig.9part C).4.DiscussionIn vivo data regarding the osteogenic potential of adipose mesenchymal stem cells (ASC)to heal bone defects remain scarce [21–24].These cells are enormously attractive for clinical use because of their ready availability and easy accessibility.Prior to clinical use,however,it must be proven that their osteogenic potential is not inferior to that of bone marrow cells.At the moment,there are few experimental studies using bone defect models addressing this issue [22,25,26].These studies suggest that undifferentiated ASC lead to functionally inferior regenerated tissues compared to MSC derived from bone marrow or periost.With the exception of the Peterson study,which examined the efficacy of BMP-2transfected ASC,the results of these studies are of only limited relevance for various reasons.One problem was that in the study of Hui et al.the cells were applied within a fibrin glue suspension [25].From our perspective,this approach is limited concerning its lack of biomechanical stability which is needed in a weight-bearing bone defect.Another problem is that a rabbit model which has a high rate of spontaneous healing was employed,and furthermore the defects were no critical size bone defects,but rather partial defects only [25].In the study by Hayashi et al.a smallanimal model (rat defect model)was used,which only allows a very limited extrapolation of results to the human patients situation and therefore must be regarded inferior to a defect model in a large animal [26].Nonetheless,these studies alert the reader to possible problems which could arise with the use of adipose stem cells and thereby justify the separate and comparative analysis of the regeneration potential of these cells with autogenous BMSC.The in vivo bone defect model studies also showed that the initially comparative studies,which demonstrated essentially identical properties of adipose and bone marrow stem cells at the in vitro level [27,28]and after heterotopic in vivo transplantation [29],cannot uncritically be transferred to the regeneration capacity of orthotopicbone.Fig.4.Quantitative evaluation of newly formed bone in the defect (in %),with regard to differences compared to ‘‘day 0’’in dependence of group assignment.Percentage of defect filled with bone in %is visualized from day 0(left column)to week 26(right column)of every group.Significances (p <0.05)compared to day 0are visualized by blackcolumns.Fig.5.Representative histological slides of animals assigned to different treatment groups (overview).P.Niemeyer et al./Biomaterials 31(2010)3572–35793576。
