血液成分与骨组织的内在联系及组织工程学评价
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摘要
研究背景及目的
骨组织工程是模拟体内生理性骨再生的过程,血液系统与骨发生发育有着分子信号机制上的联系,对两者分子机制的深入探讨有助于推动骨组织工程的发展。G蛋白偶联受体(GPCR)是细胞接受外界信号并传给胞内转录因子的第一闸门,通过转录因子调节各种细胞生物学功能。G蛋白偶联受体48(GPR48)基因敲除小鼠胚体除了表现出明显的骨骼发育迟滞外,还有贫血迹象,这种表型提示骨骼和造血之间存在某种分子联系,但这种详细的信号机制目前并不清楚。因此研究GPR48信号通路在造血和骨骼发育之间的分子联系,有助于对骨再生机制有更好的理解。
近些年来,血液中不同成分,尤其是血小板成分,在骨再生修复过程中所发挥的重要作用也引起越来越多的关注。血小板被凝血机制激活后,可释放多种生长因子,调控包括成骨细胞和破骨细胞等各种细胞的增殖分化,高效调节修复初期的骨再生。血小板这种功能的激活和维持,有赖于GPCR经多种信号转导的正常调控,因此,通过GPCR信号通路的分子联络,血小板构成了血液系统和骨再生发育之间的重要桥梁之一。新近研究的血小板裂解液(PL)是血小板衍生物,承载大量生长因子,抗原物质少,制备容易,具有高效丝裂原效应,可能成为用于骨组织工程中生长因子的重要来源。
本研究目的旨在:1、探讨血液成分和骨组织之间的分子信号联络:GPR48基因失活小鼠在胚胎红系造血和骨发育调节中的共同分子信号通路;2、将血邪辶呀庖鹤魑ひ蜃釉靥?用于骨组织工程学方法,评价其在体内外对骨再生的影响。
材料和方法
1胚胎红系造血和骨发育的共同分子信号通路研究
1.1 GPR48基因靶向灭活小鼠品系的建立、基因型的确定及表达
利用基因分泌捕获方法建立GPR48基因靶向灭活的小鼠品系(129×C57BL/6),将GPR48杂合体雌雄小鼠交配饲养繁殖获得纯合突变小鼠子体,取孕中期12.5-14.5天(E12.5-E14.5)的杂合体雌鼠剖离胚胎做相关研究。
提取胎鼠尾DNA用PCR方法鉴定胚胎的基因型,区分GPR48野生型(WT,+/+)、杂合突变型(HE,+/-)和纯合突变型(HO,-/-)小鼠。用LacZ组织特殊染色观察GPR48在E14.5 WT、HO小鼠骨骼和肝脏的表达差异。
1.2体征观察
观察E12.5-E14.5胎鼠胚体和肝脏的体态大小、颜色、活动等一般情况。
1.3外周血红细胞及血红蛋白分析
制E12.5-E14.5外周血涂片,Wright-Giemsa染色,光镜下观察红细胞形态变化。对E13.5(WT 8只,HO 7只)和E14.5(WT 5只,HO 4只)血涂片,每只取3张血片,每张血片随取3个高倍视野,高倍镜(×400)计数单个视野的有核红细胞、无核红细胞及红细胞总数,计算有核红细胞在红细胞总数中所占比例,比较统计学差异;Real-time PCR检测E13.5 WT和HO各6例胎鼠外周血中βh1和β血红蛋白链的mRNA相对表达水平,比较统计学差异。
1.4 RT-PCR和Western-blotting对胎肝和骨骼ATF4表达分析
提取E13.5 WT和HO小鼠胎肝和肋骨笼的RNA和总蛋白,分别用RT-PCR和Western-blotting检测两种小鼠胎肝和骨骼中ATF4在mRNA和蛋白水平的表达差异。
1.5组织学及免疫组化增殖分析
取E13.5胎鼠的肝脏,常规10%中性福尔马林固定,乙醇逐级脱水,二甲苯透明,石蜡包埋,切取5μm切片,HE染色,光镜观察。按照相应的操作说明作胎肝(E13.5)和骨组织(E14.5股骨髁)的免疫组化分析,PCNA试剂盒用于增殖分析,ABC法用于ATF4的免疫组化分析,用苏木素复染。
1.6统计学处理
数据用均数±标准差表示,用SPSS11.5软件包做统计学分析,采用两个独立样本的非参数检验方法进行比较,P＜0.05为统计学有显著性差异。
2用组织工程学方法评价PL的生物学效应
2.1血小板裂解液对大鼠骨髓间充质干细胞生长和成骨分化的影响
2.1.1 BMSCs的分离、培养传代和表型鉴定
成年健康清洁级Wistar大鼠8只,体重250-300g,雌雄不限。采用全骨髓分离培养法培养扩增骨髓细胞,隔日半量换液,按1:3比例传代。倒置相差显微镜观察细胞生长。取第5代细胞,进行流式细胞仪检测细胞BMSCs表面特征抗原CD45/CD90/CD29的表达。
2.12 PL制备及生长因子含量测定
取16只大鼠,采用3次离心结合反复冻融法制备PL,0.22μm无菌滤膜过滤。取6只大鼠PL样品,按照ELISA试剂盒操作说明操作,测取PL中PDGF、TGF-β1、IGF-1和VEGF含量。
2.13 PL对细胞生长能力的影响
配制含PL终浓度为5%和1%(体积分数)两种不同的条件培养基。取第5代细胞,分A1(基础培养基中含5%PL)、B1(基础培养基中含1%PL)和C1(基础培养基)三组培养,用CASY细胞分析仪测定平均活细胞数,连续培养9天,每组每天测3孔,绘制细胞生长曲线,统计学分析不同浓度PL条件下细胞生长趋势和第9天时各组活细胞数的差异。
2.14 PL对BMSCs成骨诱导分化的干预效应
2.1.4.1建立成骨诱导体系取第4代细胞,以含有0.1μmol/L地塞米松、50μg/ml抗坏血酸、10mmol/Lβ-磷酸甘油钠及10%胎牛血清的H-DMEM为基础诱导液,配制含PL终浓度为5%和1%(体积分数)两种不同的条件诱导培养基,分别用于A2、B2组细胞的成骨诱导,C2组为不含PL的基础诱导液作为对照。连续培养20天,倒置相差显微镜下连续动态观察细胞形态变化和生长状况。
2.1.4.2不同PL诱导分化条件下BMSCs的ALP(碱性磷酸酶)活性和矿化形成
诱导7天时,各组细胞作ALP染色,观察胞浆内颗粒情况;诱导2、8和12天时,测取各组3个样本的ALP和TP(总蛋白)含量,以ALP/TP比值作为ALP活性定量指标。
诱导20天时,对细胞行Von Kossa染色和Alizarin red染色,观察细胞外基质矿化形成。对Alizarin red染色,每组取3例,每例随取5个视野,用图像分析软件计数单个视野中(×200)矿化结节数量和面积,比较各组差异。
2.2 PL/ADBG/CG与BMSCs的生物相容性研究
2.2.1同种异体脱钙骨颗粒(ADBG)的制备
大鼠10只(同上述),取四肢骨(包括干骺端),经深冻、超声清洗、粉碎后筛取直径300-500μm大小的骨粒,于超声条件下0.6N盐酸洗脱、冻干辐照。
2.2.2Ⅰ型胶原(CG)与ADBG的双相沉降复合
用0.1M乙酸作溶剂将CG配制成质量体积分数为0.25%的胶原溶液,辐照灭菌,4℃条件下将ADBG按质量体积分数为6%的比例加入CG溶液中,搅匀形成混悬液,加1N NaOH将混悬液的PH值调整到7.0左右,这时发生CG和ADBG发生共同沉降粘结,形成凝胶海绵样复合物,CG/ADBG的质量分数约为100mg/1g,取出后用无菌滤纸吸干,-20℃储存备用。
2.2.3 PL/ADBG/CG制备、复合培养
按5mL PL与5g ADBG/CG比例将两者在4℃无菌条件下浸泡24小时,大约60%PL被吸附于CG/ADBG。取PL/ADBG/CG复合物约100mg置于24孔培养板底部,与第4代BMSCs复合培养7d,倒置显微镜、电镜(3d)观察细胞形态及粘附生长状况。
2.3体内移植评价PL的生物学效应
2.3.1股骨髁缺损造模及材料移植
取大鼠(同上述)30只,分A、B、C三组,每组10只/20侧。无菌条件下制取大鼠双侧股骨髁内60-70%的腔隙性缺损。A、B和C组分别填塞植入等量(大约500mg/缺损)PL/ADBG/CG、ADBG/CG和CG。
2.3.2术后缺损修复效果评价指标
2.3.2.1 X线及骨密度计量分析
术后1-4周时,摄X光片作放射线检查了解新骨生长和缺损修复情况;用Image-Pro Plus 5.0图像分析软件测取2、4周缺损修复区单位面积的灰度值,每组测8个股骨髁,以代替骨密度评价缺损区骨修复状况。
2.3.2.2组织学观察及形态计量学分析
术后4周,每组随取3只动物,截取双侧股骨髁,经固定、脱钙、脱水、等常规病理操作方法,制作厚度5μm石蜡切片。常规HE染色,光镜观察。每组随取3张切片,每张切片随选5个视野(×200倍),用Image-Pro Plus 5.0图像分析软件测取缺损修复区单个视野下成骨区面积(以总像素数表示),比较修复效果。
2.3.2.3抗压生物力学测试
A、B、C每组随取实验动物3只,截取股骨髁,另取6具同窝大鼠正常股骨髁做对照D组。将股骨髁平置于载物台,压力装置以5mm/min的加载速度施压,电脑记录股骨髁位移达2mm时的破断载荷,并计算破断强度。
2.3.2.4三色流式细胞术测定外周血T-淋巴细胞亚群
4周处死动物前,A、B和C组各随选3只动物,另取三只同窝正常大鼠作为对照,麻醉后自心内采血2mL,利用三色流式CD4-FITC,CD3-PE和CD8-TC,检测各亚群百分比及CD4/CD8比值。
2.4统计学处理
用SPSS11.5软件包做统计学分析,数据用均数±标准差表示,采用析因分析交互效应,对于单因素效应采用One-way ANOVA,LSD法用于组间多重比较,P＜0.05为统计学有显著性差异。
结果
1 Gpr48-cAMP-CREB-ATF4分子信号转导共同调节胚胎红系造血和骨发育
1.1 GPR48基因的靶向灭活及其在胚胎期肝脏和骨组织表达
在小鼠Gpr48基因序列的内含子1区中随机插入了一个较大的分泌型捕获载体(11.9kb)。这个载体插入引起GPR48基因在小鼠基因组中表达缺失,PCR结果所显示的条带为750bp,而不出现GPR48正常基因条带(450bp)。LacZ组织学染色显示在胚胎E13.5肝脏和骨组织内均有GPR48表达,被染成蓝色,骨骼染色呈强阳性。
1.2 GPR48-/-小鼠发育迟滞和红系造血表型
大体观察发现与WT相比,E12.5-E14.5 HO胎鼠身体发育低下,体型、肝脏均明显减小,四肢骨骼发育短小,而且胎体和肝脏的颜色较苍白,皮下血管细微不发达。外周血涂片显示HO有核红细胞相对数明显升高,高倍镜下计数结果显示,E13.5天时HO和WT胎鼠的的有核红细胞在总红细胞数中所占的比例分别为(79.91±3.57)%和(30.00±2.17)%,HO比WT提高了约2.7倍水平,两者间比较,z=-3.24,P＜0.01,差异显著有意义;E14.5天时HO和WT分别为(54.99±1.93)%和(27.15±1.88)%,两者间比较,z=-2.45,P＜0.02,差异显著有意义。血红蛋白链Real-Time PCR分析结果:βh1链相对含量(WT为47.17±13.30,HO为113.50±23.44),β链相对含量(WT为136.50±20.42,HO为62.8±16.13)。与WT相比,HO胚胎血红蛋白βh1链mRNA相对量升高(z=-2.882,P＜0.01,n=6),差异有显著性意义;而成熟血红蛋白β的mRNA相对表达量则下调(z=-2.882,P＜0.01,n=6),差异有显著性意义。
1.3 ATF4在胎肝和骨中的表达分析
胎肝和骨RT-PCR结果显示,HO胎鼠ATF4在约400bp处出现的条带亮度明显弱于WT对照;Western-blotting结果同样显示在约40kD处,HO出现的条带亮度明显弱于WT对照。免疫组化分析显示,ATF在HO胎鼠肝脏和骨组织中的表达呈现弱阳性,显著低于WT中ATF4的表达。
1.4组织学及增殖分析
肝脏HE染色结果显示,纯合体与野生型相比,肝细胞形态类似,但定向红系祖细胞和红细胞岛明显减少。PCNA增殖分析结果显示HO胎肝(E13.5)和骨组织(E14.5股骨髁)呈现弱阳性染色,处于增殖期的细胞明显少于WT,E14.5HO股骨皮质骨厚度也明显减少。
2用组织工程学方法评价PL的生物学效应
2.1 PL对大鼠体外BMSCs生长和成骨分化的影响
2.1.1 BMSCs的分离、培养、传代和表型鉴定
BMSCs经分离培养传至第5代时即纯化为单一纺锤状,规则平行排列,类似于成纤维细胞形态。流式细胞仪分析,这些细胞群落表达的BMSCs特征表面抗原高度均一,呈现CD45~-CD90~+CD29~+,符合BMSCs的表型特征。
2.1.2 PL中生长因子含量
经ELISA定量分析,PL中PDGF、TGF-β1、IGF-1和VEGF浓度分别为405±59.58、140±26.65、85.83±16.86和82.5±11.73(pg/mL)。
2.1.3不同浓度PL对BMSCs增殖的影响结果
生长曲线显示自第3天开始细胞生长迅速,随着PL浓度的提高和时间延长,各观察点活细胞数呈现递升趋势;A1组细胞增殖较快,在第3天达到对数生长期,比C1组提前约1天。析因分析结果显示,不同浓度的PL对BMSCs的增殖差异有显著性意义(F=320.33,P＜0.01),培养时间对BMSCs的增殖差异也有显著性意义(F=406.04,P＜0.01),PL浓度和培养天数之间有交互效应(F=36.25,P＜0.01),说明随着PL浓度的升高和培养时间的延长细胞增殖呈上升趋势。进一步用TUkey HSD法做差异性检验的多重比较,结果显示A1组对细胞增殖的影响高于B1(P＜0.01)、C1组(P＜0.01),B1组对细胞增殖的影响高于C1组(P＜0.01),差异有显著性意义。
第9天时各组活细胞数分别为A1(988436±15720.42)个、B1(616666.67±12423.1)个和C1(345000±21931.71)个,单因素方差分析结果显示各组间差异有显著性意义(F=1064.11,P＜0.01);进一步组间多重LSD比较显示,A1组活细胞数高于B1(P＜0.01)组和C1组(P＜0.01),B1组高于C1组(P＜0.01),差异有显著性意义,其中细胞数达到C1组的近3倍水平。
2.1.4不同PL条件下对BMSCs成骨诱导分化的影响
2.1.4.1不同诱导条件下对BMSCs ALP活性的影响
不同PL条件成骨诱导分化下,C2组细胞长突起逐渐变短,胞体变宽、变大,7d时胞浆内出现较多ALP染色阳性的粗大颗粒。B2组与C2组细胞的形态改变相类似,但程度不如A2组明显,而且细胞增殖较C2组快。A2组细胞生长较B2、C2迅速,细胞形态改变不如B2、C2两组显著,胞浆颗粒不发达,细胞处于迅速增殖状态。20天时,B2和C2组细胞外有大量矿物沉积;A2组细胞仍呈密集生长,排列杂乱,细胞外基质中沉积物明显少于B2、C2两组。
ALP活性定量分析中,第2、8、12天A2、B2、C2组ALP活性析因分析显示,不同诱导条件下ALP活性的差异有显著性意义(F=45.698,P＜0.01);进一步组间LSD多重比较显示,P_(A2-B2)＜0.01,P_(A2-C2)＜0.01,差异有显著性意义,P_(B2-C2)＞0.05。不同培养天数间差异有显著性意义(F=121,P＜0.01),不同的PL浓度与天数对ALP活性的影响存在显著性交互效应(F=6.86,P＜0.01),以C2组在第12天时的ALP活性最高。
2.1.4.2不同诱导条件下对BMSCs基质矿化的影响
B2、C2组细胞在20天时在胞外基质中出现大量沉积物,而A2组20天时细胞仍密集生长,胞外基质中沉积物较少。Von Kossa染色和Alizarin red染色显示A2两组矿化沉积明显少于B2、C2组。Alizarin red染色单个视野(×200倍图像)中各组矿化结节计数A2、B2和C2组分别为7.67±1.10、14.0±2.23和14.97±1.88(个);组间方差分析显示F=14.593,P＜0.01,差异有显著性意义;进一步组间LSD两两比较,A2组矿化结节数显著低于B2(P＜0.01)和C2(P＜0.01),差异有显著性意义,而B2与C2间无显著性差异(P＞0.05)。
A2、B2和C2矿化结节的面积分别为161778.73±44550.80、337349.67±56083.24和415921.73±71725.39(像素),组间方差分析显示F=14.831,P＜0.01;进一步组间LSD两两比较,A2组钙结节面积显著低于B2(P＜0.01)和C2(P＜0.01),差异有显著性意义,而B2与C2间无显著性差异(P＞0.05)。
2.2 PL/BDG/CG与BMSCs体外共育的生物相容性
共育3、7d,倒置相差显微镜显示BMSCs细胞形态无明显异常,材料边缘及周围可见大量BMSCs生长。扫描电镜显示细胞呈长梭形,有多个长突起,紧密贴附于材料表面,融合呈网状连接,有足突状突起向材料内部深入生长。
2.3 PL/ADBG/CG体内移植对股骨髁缺损修复重建效果
2.3.1放射学及骨密度计量学分析
A组,1周时缺损修复区可见少量稀疏絮状阻射影;2周时絮状阻射影密度较前增加;3周时阻射影质地渐趋均匀、致密;4周时缺损中心区阻射影密度接近正常骨质,缺损基本修复。B组,1周时缺损中心区为大量透亮区;2周时阻射影密度较前增加;3周时仍有较多不规则的透亮区;4周时阻射影密度趋于致密,仍有少量不规则的透亮区。C组,1周时缺损仍为明显的透亮区;2周时,A组缺损区有少量模糊阻射影;3周时中心区仍以大量透光区为主;4周时缺损未见修复迹象。
灰度(代骨密度)计量分析显示,2周时,组间方差分析F=25.08,P＜0.01,各组灰度值间差异有显著性。进一步组间LSD多重比较,A组单位面积的灰度值高于B组(P＜0.01)和C组(P＜0.01),B组高于C组(P＜0.05),差异有显著性意义。4周时方差分析F=29.08,P＜0.01,各组灰度值间差异有显著性。进一步组间多重比较,A组单位面积的灰度值高于B组(P＜0.01)和C组(P＜0.01),B组高于C组(P＜0.05),差异有统计学意义。
2.3.2组织学及组织计量学分析
4周时,A组修复区可见已成活骨片,有较多成骨细胞和新生类骨质长入,也可见正在被吸收的骨片,周围包裹大量纤维组织,伴少量炎细胞浸润;正常骨端向缺损区有大量新生骨延伸和多处软骨内成骨现象,成骨和破骨活动均较B和C组活跃。B组仍有较多死骨存在,可见崩解的碎片和正在被吸收的骨颗粒,有少量骨颗粒成活,但成骨和破骨现象不如A组活跃。C组仍填充以大量纤维组织,周围多处正常骨壁成骨不活跃。
经骨组织形态计量学分析,对单个视野(×200倍)中其新生骨软骨和成活的移植骨片总面积(像素值)作方差分析,F=122.398,P＜0.01,差异有显著性意义;进一步作组间两两多重比较,结果显示A组成骨面积高于B组(P＜0.01)和C组(P＜0.01),B组高于C组(P＜0.01),差异有有显著性意义。
2.3.3抗压生物力学评价
各组标本经抗压生物力学测试,对破断载荷和破断强度做方差分析,F=64.469,P＜0.01,差异有显著性意义;进一步作组间两两多重比较,结果显示A组破断载荷和破断能量均高于B(P＜0.01)和C组(P＜0.01),但低于正常对照D组(P＜0.01),B组显著高于C组(P＜0.01),C组最低,差异有显著学意义。
2.3.4三色流式细胞术T细胞亚群测定
三色流式细胞术分析结果见表2.3.3,经方差分析,CD3~+CD4~+CD8~-、CD3~+CD8~+CD4~-、CD4/CD8在各组间均无显著性差异(P＞0.05)。
结论
血液成分(包括红细胞和血小板)与骨组织的发育、再生是存在内在联系的,血小板裂解液可替代多种生长因子应用于骨组织工程方法。
1.GPR48-cAMP-CREB-ATF4分子信号转导是红系造血和骨发育共同的转导信号控制通路之一,同时调控小鼠胚胎期红细胞的成熟和骨发育,构成连通血液系统和骨骼组织的分子信号桥梁。GPR48的灭活同时造成红系造血和骨发育缺陷,这对解释临床上某些不明原因的疾病具有潜在的参考价值。
2.GPR48信号通路通过调节细胞增殖影响骨和红细胞发育。
3.PL由血小板衍生而来,是多种生长因子的承载体系,可参与骨形成调控。PL以剂量依赖方式,对大鼠BMSCs发挥丝裂原作用,促进细胞增殖;而对BMSCs成骨诱导分化过程中的ALP活性和矿化形成呈现剂量依赖性的抑制作用。PL对BMSCs增殖和分化的不一致效应可能对早期骨修复有利。
4.PL可作为生长因子用于骨组织工程学方法,所构建的重组合异体脱钙颗粒骨PL/ADBG/CG与BMSCs有良好的生物相容性,复合移植后可促进骨缺损的早期修复过程。
5.PL对骨重建有促进作用,可能与多因子协同作用下激发骨吸收-骨形成偶联机制有关。
Background and purposes of the research
Bone tissue engineering refers to a process to mimic physiological bone regeneration. The connection of molecular signal mechanism exists between blood system and skeleton, on which performing profound research helps to the further development in bone tissue engineering. G protein coupled receptors (GPCRs) represent the first switch not only to receive signals from outside, but also to transduct molecular signals to intracellular transcript factors which function to regulate the biological activities of various cells. G protein coupled receptor 48 (GPR48) gene knock-out fetal mice showed both anemia appearance and marked development retardation of skeleton, this phenotype suggested that some molecular connection existed between hematopoiesis and skeleton, but we haven't made it clear. Therefore, it is helpful to obtain profound acknowledge about bone regeneration under the research on the molecular connection between hematopoiesis and skeleton development through GPR48 signal pathway.
In recent years, varied components in blood, especially platelet, have attracted more and more attention due to the important role they played in bone regeneration. Blood platelets function to regulate the proliferation and differentiation of varied cells including osteoblast and osteoclast by way of releasing various growth factors when activated by coagulate mechanism. The maintenance and activation of normal platelets' functions depends on the regulation by many signal pathways via GPCRs. Therefore blood platelet forms the bridge to connect blood system with bone repair through GPCRs pathway. Most resently, platelet lysate (PL), which has been reported to be derived from platelet and loaded with large amount of growth factors and less antigen, functions as a highly effective mitogen, all of which implies that PL might be a potential resource of growth factor substitutes applied into bone tissue engineering.
The purpose of this reaseach aims: 1) To explore the molecular connection between blood components and skeleton tissue: the shared molecular signal pathway in erythropoiesis and skeleton development for GPR48 gene-traped fetal mice; 2) To evaluate the effects of PL as a resorce of growth factors on bone regeneration by way of bone tissue engineering strategies both in vitro and in vivo.
Material and Methods
1 Study on the shared molecular signal pathway functions both in fetal erythropoiesis and skeleton development
1.1 Generation of GPR48 knocked out mice, determination of genotype and expression pattern
Gpr48 knocked out mice of (129×C57BL/6) were generated via secretory-trap approach. Homozygous were obscured by intercrossing heterozygous mice. E12.5～14.5 fetal mice isolated from HE female pregnant mice were selected for this research program.
Genotype of mice (wild-type +/+, heterozygous +/-, and homozygous -/-) was determined by PCR to distinguish the type of GPR48 genomic DNA extracted from the tails of fetal mice. LacZ histochemistry assay was carried out to detect the discrepancy of GPR48 expression in skeleton and liver between GPR48 wild-type and homozygous fetal mice.
1.2 Grossic phenotype
To observe the gross characteristics including size, color both for embryos and liver, the size of liver and action status of fetal mice obscured from E12.5～14.5 embryos.
1.3 Analysis of erythrocytes and hemoglobin chains in peripheral blood
Blood smears were prepared by wedge technique following Wright-Giemsa staining protocol for E12.5～14.5 embryos. For E13.5 fetal mice (8 WT and 7 HO) and E14.5(5WT, 4HO), 3 blood smear slides were randomly selected from each embryo in WT or HO, and 3 high power views of each slide were randomly chosen to count the number of nucleated erythrocytes and enucleated erythrocytes, and calculated the relative level of nucleated erythrocyte in total number of erythrocytes for comparisons.
Real-time PCR was performed to detect the mRNA level ofβh1 andβhemoglobin chains in blood of E13.5 WT (6 embryos) and HO (6 embryos).
1.4 ATF4 expression in fetal liver and skeleton
The total RNA and protein extracted from liver and bone (lib cage) of both E13.5 wild-type and homozygous embryos were detected by RT-PCR and Western-blotting approaches respectively to assay ATF4 expression in liver and skeleton (lib cage) at both of mRNA and protein level.
1.5 Histology and Immunohistochemistry (IHC) assay
Fetal liver (E13.5) dissected from mice were fixed in 10% formalin and embedded in paraffin according to the standard techniques. Five micrometer-thick sections were stained with hematoxylin and eosin (H&E). For proliferation assays, the sections of both the liver (E13.5) and femur (E14.5) were stained with anti-PCNA using PCNA kit and then counterstained with hematoxylin. For ATF4 expression in liver and bone, the following antibodies were used: rabbit polyclonal anti-ATF4 (C-20) (1:100), hematoxylin was carried out as counterstain.
1.6 Statistical analysis
Data were presented as mean±standard deviation and analyzed by 2-independed samples non-parameteric test with SPSS 11.5 software. For all analyses, P < 0.05 was considered statistically significant.
2 Evaluation of the biological effects of PL via the strategies of tissue engineering
2.1 Effects of PL on the proliferation and osteogenic differentiation of BMSCs.
2.1.1 Isolation, expansion and phenotypic analysis of BMSCs
Total 8 adult healthy Wistar rats with weight of 250-300g of clean grade were used in this section, whole bone marrow was cultured and expanded by changing medium in half every other day and passaged at a ratio of 1:3. The cell growth was observed by inverted microscope. The 5th passages were prepared to determine the expression of cell surface antigens CD45/CD90/CD29 which function as the specific marker of BMSCs by flow cytometry.
2.1.2 Preparation of PL and ELISA assay for the determination of growth factors
PL was obtained through three times of centrifugations and repeated freeze-thaw for the venous blood aspirated from 16 rats and filtrated through 0.22μm sterilized filter membrane. ELISA assay was conducted following the manufacture's protocol to determine the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL samples from 6 rats.
2.1.3 Effects of PL on cell growth
PL at final concentration of 1% and 5% in basic medium (V/V) was prepared as conditioned medium. BMSCs of the 5th passages were classified into three groups of A1 (5% PL in basic medium) , Bl(l% PL in basic medium), and C1(no presence of PL in basic medium as controls) to expand BMSCs respectively for a series of 9 days so as to draw the cell growing curves through the assay by CASY cell analyzer. The living cell number of all groups was compared at 9d.
2.1.4 Effects of PL on osteogenic differentiation of BMSCs
2.1.4.1 The establishment of osteogenic differentiation system
The cells at fourth passage were performed for osteogenic differentiation under induction environment in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as controls). The morphological changes of cells were dynamically observed with inverted phase contrast microscope.
2.1.4.2 ALP (Alkaline phosphatase) activity and mineral formation of BMSCs under inductive differentiation with different PL concentration
ALP staining (7d) and ALP/TP (2, 8,12d) of the cells were detected to evaluate ALP activity for 3 samples of each group. At 20d, Von Kossa and Alizarin red staining were both carried out to observe extracellular mineral formation. For Alizarin red staining, 3 cases of each group were used to examine the mineral formation in extracellular martrix, randomly chose 3 views (×200) to obtain the number and area of mineralized nudes under microscope for each case with image analyis software.
2.2 Study on the biocompatibility between PL/ADBG/CG and BMSCs
2.2.1 Preparation of allogeneic decalcified bone granules (ADBG)
10 Wistar rats were used to prepare ADBG, the limbs bone were processed through deep freeze, supersonic clean, grinding to obtain granules with diameter range of 300-500μm. The granules were further processed through 0. 6N HCl washing, lyophilization and radiation.
2.2.2 The combination of Collgen I (CG) with ADBG via dural-phase precipitation
The CG resolution at the concentration of 0.25% (W/V) was prepared using 0.1M acetic acid as basic solvent and sterilized by radiation. ADBG was added into this CG resolution according to W/V fraction of 6% and mixed well to obtain a suspension, to which IN NaOH was added to adjust the PH value to 7.0 approximately, then CG and ADBG precipitated and adhered together to form a gel-like composite CG/ADBG (100mg/1g), which then was dried on the filter paper and stored at -20℃.
2.2.3 Preparation of PL/ADBG/CG and co-culture with BMSCs
PL/ADBG/CG was obtained by immersing 5g ADBG/CG with 5mL PL under aseptic condition at 4℃. BMSCs of fifth passage were coincubated with PL/ADBG/CG for 7d to observe the biocompatibility and adhensive growth with inverted microscope, and scanning electron microscope was conducted for the composite at 3d.
2.3 Effects of PL/ADBG/CG transplantation on reconstruction of femoral condylar defect in vivo
2.3.1 Establishment of femoral condylar defect model and graft transplantation
30 Wistar rats as above description were devided into 3 groups (A, B, C) with 10 rats/20 condyles in each group. The bilateral femoral condylar defects (with 60-70% bone loss) were established for these animals and transplanted with 500mg of A(PL/ADBG/ CG), B(ADBG/CG) or C(CG) filled into defect respectively.
2.3.2 Evaluation on bone defect repair after operation
2.3.2.1 X-ray and bone density assay
After operation, X-ray was performed to observe the new bone formation and repairing status. Image-Pro Plus 5.0 software was used to measure the gray level per unit area in repairing field of 8 femoral condyls in each group at postoprerative 2 and 4w, which represented the bone density for the evaluation on bone defect repair.
2.3.2.2 Histology and morphological quantitative analyses
At 4 weeks, the femoral condyles of 3 rats in each group were cut off and processed by fixation, decalcified, dehydration, and other procedures to make paraffin sections with thickness of 5μrn. After regular HE staining, microscope was conducted to observe the bone formation, 3 sections were randomly selected and 5 views in each section under microscope (×200) were randomly chosen to measure the new bone area (pixels) per view (×200) with Image-Pro Plus 5.0 software.
2.3.2.3 Biomechanical assay of anti-pressure
At 4 weeks, 6 condyles from 3 rats in each group were used for mechanical test together with 6 normal condyles of littermates as group D. Condyles were horizontally placed on the object stage, and pressed at the loading speed of 5mm/min. The destructive loading was recorded when displacement attained to 2mm and calculated the destructive intensity.
2.3.2.4 Determination of T lymphocyte subsets in peripheral blood
Before killing rats at 4w, 3 rats were randomly choosed from A、B and C ,and another 3 rats littermates were performed as controls. Three-color flow cytometry was conducted by using CD4-FITC, CD3-PE and CD8-TC to detect percentage of T lymphocyte subsets as well as the ratio of CD4/CD8.
2.4 Statistical analysis
Data were presented as mean±standard deviation and analyzed by Factorial analysis for interaction effects, and One-way ANOVA was performed for one-factor effect. LSD was conducted for related multiple comparisons. For all analyses, P<0.05 was considered statistically significant.
Results
1 GPR48-CAMP-CREB-ATF4 signal transduction referred to the shared molecular signal pathway to regulate the development of both erythropoiesis and skeleton at embryonic stage
1.1 Targeted inactivation of GPR48 and expression pattern in fetal liver and skeleton
The murine GPR 48 genomics was disrupted by randomly inserting a large secretory trap vector (11.98kb) to the intron 1. The insertion caused deletion of GPR48 in mouse genome, which was confirmed by PCR analysis which showed that GPR48-/- null mice didn't present the band of normal GPR48 gene at 450bp. LacZ staining showed that GRP48 was expressed both in E13.5 fetal liver and skeleton in blue color.
1.2 Growth retardation and erythropoiesis phenotype
The gross appearance of GPR48-/- null fetal mice from E12.5 to E14.5 showed growth retardation both in body size and liver size, limbs were shorter and both body and liver were markedly paler when compared with wild-type controls.
The peripheral blood smear for E13.5～E14.5 indicated that HO had more nucleated erythrocytes in blood. E13.5 blood smear assay displayed the proportion of nucleated erythrocyte in total erythrocytes of GPR48-/- null fetal mice elevated about 2.7 folds of normal litters, which with significant difference between these two groups (HO 79.91±3.57, WT 30. 00±2. 17; z=-3.24, P<0.01). For E14.5 mice, there also was markedly difference between HO and WT (HO 54.99±1.93, WT 27.15±1.88; z=-2.45, P<0.02).
Real-time PCR showed that relative mRNA level ofβh1 in E13.5 homozygous blood was significantly increased than that in normal littermates (z=-2.882, P<0.01). Otherwise, the relative mRNA level ofβin homozygous blood was markedly reduced than that in wild-type littermates (z=-2.882, P<0.01).
1.3 ATF4 expression in GPR48-/- fetal liver and skeleton
In RT-PCR, ATF4 expression in E13.5 GPR48-/- fetal liver and skeleton (lib cage) showed that HO presented a significantly weaker band at 400bp than WT. In Western-blotting, HO also presented a markedly weaker band at 40kD than WT. Immunohistochemistry assay indicated that ATF4 expression in both HO liver (E13.5) and skeleton (E14.5 femoral condyle) was weakerly in positive staining than normal controls.
1.4 Histology and proliferation assay
Histological examination and hematoxylin and eosin (H&E) staining showed that the gross morphologic features of homozygous fetal livers were normal in cellular architecture. However, fewer erythroid precursor or definitive progenitors and erythroid foci were visible in the homozygous fetal livers from E13.5 embryos compared with that in wild-type.
Immunohistochemical assays (IHC) of PCNA for proliferation assay indicated that the thickness of femoral cortex in E14.5 HO was significantly thinner than that in WT control. The strength of proliferating stain was markedly weaker in the fetal livers and femoral condyles of homozygous Gpr48 null mice compared with their control littermates.
2 Evaluation of the biological effects of PL via the strategies of tissue engineering
2.1 Effects of PL on proliferation and osteogenic differentiation of BMSCs
2.1.1 Isolation, expansion and phenotypic analysis of BMSCs
Under inverted microscope, cells at 5th passage presented the similar morphology of uniform long-spindle shape as fibroblasts and in regular arrangement of parallel aggregation. The cells expressed CD45~-CD90~+CD29~+ antigens by assay of flow cytometry which conformed to the characterized cell surface antigens of BMSCs.
2.1.2 ELISA assay of the concentration of growth factors in PL
ELISA assay presented that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL attained to 405±59.6, 140±26.65, 85.8±16.86, and 82.5±11.7 (pg/ml) respectively.
2.1.3 Effects of PL on cell proliferation
Under different conditioned medium, BMSCs in A1, Bl and C1 displayed nearly the same configuration with long shuttle-shape as fibroblasts. The growth curve displayed that cells grew faster from 3d and the living cells number at every timepoint presented ascending trend along with the prolongation of culture time. Cells in A1 grew fastest within these 3 groups from 3d into logarithmic growth phase which was 1 day earlier than that in Cl. The results of Factional analysis showed that different PL concentration had significant difference in effects on the cell proliferation (F=320.33, P<0.01), while different culture time also markedly affect the cell proliferation (F=406.04, P<0.01), and there was interaction effect between different PL concentration and cultural time (F=36.25, P<0.01) which certificated that cell proliferation presented ascending trend along with the prolongation of culture time in different PL concentration. Further LSD assay of the multiple comparations presented that cell proliferation of group A1 was higher that that of B1 (P <0.01) and C1(P <0.01), and that B1 was higher than C1(P <0.01), these differences were significantly different.
At 9d, the living cell number in A1, B1 and C1 was 988436±15720.42, 616666.67±12423.1, and 345000±21931.71 respectively, One-way ANOVA showed there was significant difference within these groups (F=1064.11, P<0.01). Further multiple comparisons with LSD showed that the living cell number in A1 was significantly higher compared with that in B1 (P <0.01) or C1(P <0.01), and living cell number in B1 was higher than that in C1(P <0.01), there differences were significant within them. The living cell number in A1 reached about 3 folds of that in C1(P<0.01) at 9d.
2.1.4 Effects of PL on the osteogenic differentiation of BMSCs
For differentiation analysis, morphological observation displayed BMSCs in group A2 showed slower shape-changes but higher proliferation than that in group B2 or C2. Cells in B2 showed higher proliveration than that in C2 but lower that that in A2. At 7d, the cells in group A2 showed smaller amount of granules with ALP positive staining in cytoplasm when compared with B2 or C2. Moreover, at the day of 20, the cells in group A2 still displayed much higher dense growth.
At the day of 2, 8, and 12, Factorial assay of ALP activity showed there was marked diffence within these groups (F=45.698, P<0.01). Further multiple comparisons showed marked reduce of ALP activity in A2 compared with that in B2 (P <0.01) or C2 (P <0.01) and B2 displayed no significant decrease compared with C2 (P >0.05). Different days showed significant difference to affect the ALP activity (F=121, P<0.01). There was interaction effect between different days and different PL concentration (F=6.86, P<0.01) , the highest value of ALP activity occurred at 12d.
At 20d, both Von Kossa and Alizarin red staining showed there were large amount of mineral deposits occurred in the excellular martrix of B2 and C2, but smaller amount in A2 compare with B2 and C2.
In Alizarin red staining, the mineral nodes under the single view (×200) attained to A2 (7.67±1.10), B2 (14.0±2.23), C2 (14.97±1.88) respectively. With One-way ANOVA assay, there was significant difference in the nodes number under the single view within these groups (F=14.593, P<0.01), further LSD asaay showed significantly smaller amount of mineral deposits produced in the excellular martrix of A2 compared with B2 (P <0.01) or C2 (P <0.01), while no marked difference between B2 and C2 (P>0.05). The area of mineral nodes under the single view (x200) attained to A2 (161778.73±44550.80), B2 (337349.67±56083.24), C2 (415921.73±71725.39) pixels respectively. With One-way ANOVA assay, there was significant difference in the area of nodes under the single view within these groups (F=14.831, P<0.01), further LSD asaay showed significantly larger area of mineral deposits produced in the excellular martrix of A2 compared with B2 (P <0.01) or C2 (P<0.01) , while no marked difference between B2 and C2 (P>0.05).
2.2 The biocompatibility of PL/ADBG/CG with BMSCs
After 3d and 7d in coculture, BMSCs grew normally around or adhered to PL/ADBG/CG with no significant shape change under inverted microscope. Under scanning microscope at 3d, living cells with long processes tightly adhered to the surface and grew toward the deep inner of the composite.
2.3 Effects of PL/ADBG/CG transplantation on reconstruction of femoral condylar defect in vivo
2.3.1 Radiology and quantitative assay of bone density
In group A, X-ray at 1w showed small amount of resistance projective image in repairing region occurred; at 2w, the density of resistance projective image was increased than before; at 3w, the image of resistance projective gradually tended dense and homogenous; at 4w, the image of bone density was close to normal bone nearby and the defect was nearly repaired. In group B, at 1w the center of defect region showed large amount of translucent region; at 2w, the image density was increased than before; at 3w, there was more irregular translucent region; at 4w, the defect center majored large amount of translucent region and no clues of defect repair.
These observations were conformed by quantitative assay of bone density which showed that there was dramatic difference within A, B, and C (2w: F=25.08, P<0.01; 4w: F=29.048, P<0.01). Further multiple comparisons presented that at either 2 or 4 weeks, the bone density in A presented significantly higher than that in B (P <0.01) or C (P <0.01), and B was also significantly higher than C (P <0.05) in bone density.
2.3.2 Histology and quantitative assay
Histology assay at 4 weeks displayed that there were more osteoblast and new osteoid growing into bone granules, also the phenomenon of absorption was easy to be seen in the dead bone which surrounded by fibrous tissues with small amount of inflammatory cells infiltration. Group A showed large amount of new bone forming from normal bone nearby expanded and more endochondral ossification could be observed. Moreover, there were more osteoblastic and osteoclastic activities in A than in B or C. There were more dead bone pieces in disintegration and absorption and small amount of new bone formation occurred in defect region of group B. There were still large amount of fibrous tissues to fill the defect field in group C, many sites of normal bone walls showed less active in ossification.
Quantitative assay displayed the newly reparative bone area within these 3 groups was dramatically different (F=122.398, P<0.01), and further LSD multiple comparisons showed that the newly formed bone area in A was significantly higher than that in B (P <0.01) or C (P <0.01), and B showed more bone formation than C (P
2.3.3 Anti-press biomechanical evaluation
Anti-press mechanical measures showed that there was significant difference within A, B, C, and D (F=64. 469, P<0. 01). Further multiple comparisons were conducted and the results showed that either destructive load or destructive energy was higher in A than that in B (P <0.01) or C (P <0.01), but markedly lower than in D (P <0.01) which referred as normal controls, while C presented the worst result with statistic significance (P <0.01).
2.3.4 T lymphocyte subsets determination
The T lymphocyte subsets of CD3~+CD4~+CD8~-, CD3~+CD8~+CD4~- and the ratio of CD4/CD8 showed no significant difference within these 4 groups by One-Way ANOVA assay (P>0.05).
CONCLUSION
Blood components incluling erythrocytes and platelets present the internal connection with skeleton tissues either in development or bone regeneration, and platelets functioning as a resorce of multiple factors may be applied to the strategies of bone tissue engineering.
1 GPR48-cAMP-CREB-ATF4 signal transduction, which is one of the shared molecular signal pathways to regulate the development of both erythrocyte and skeleton in fetal mice, forms the molecular bridge to connect blood system with skeleton tissue.
2 GPR48 signal pathway regulates the development of both erythrogenesis and skeleton via cell proliferation.
3 PL is derived from platelet and presents to be a kind of system carrying various growth factors to regulate bone formation. PL functions as a kind of mitogen to promote the proliferation of BMSCs in a dose-dependent manner, while inhibits both of ALP activity and mineral formation of BMSCs also in dose-dependent manner under osteogenic induction environment. But this non-coordination effects of PL on proliferation and differentiation implies that PL may function to promote the bone formation at earlier stage.
4 PL presents to be a nice substitute for growth factors and can be applied to the strategy of bone tissue engineering. PL/ADBG/CG, the recombinant decalcified allograft bone, which shows better compatibility with BMSCs, can promote bone defective repair at early stage.
5 PL promotes the bone remodeling in repairing the femoral condylar defects, which may be related with the activation of coupling mechanism between bone absorption and formation via the synergistic effects of various factors.
骨组织工程是模拟体内生理性骨再生的过程,血液系统与骨发生发育有着分子信号机制上的联系,对两者分子机制的深入探讨有助于推动骨组织工程的发展。G蛋白偶联受体(GPCR)是细胞接受外界信号并传给胞内转录因子的第一闸门,通过转录因子调节各种细胞生物学功能。G蛋白偶联受体48(GPR48)基因敲除小鼠胚体除了表现出明显的骨骼发育迟滞外,还有贫血迹象,这种表型提示骨骼和造血之间存在某种分子联系,但这种详细的信号机制目前并不清楚。因此研究GPR48信号通路在造血和骨骼发育之间的分子联系,有助于对骨再生机制有更好的理解。
近些年来,血液中不同成分,尤其是血小板成分,在骨再生修复过程中所发挥的重要作用也引起越来越多的关注。血小板被凝血机制激活后,可释放多种生长因子,调控包括成骨细胞和破骨细胞等各种细胞的增殖分化,高效调节修复初期的骨再生。血小板这种功能的激活和维持,有赖于GPCR经多种信号转导的正常调控,因此,通过GPCR信号通路的分子联络,血小板构成了血液系统和骨再生发育之间的重要桥梁之一。新近研究的血小板裂解液(PL)是血小板衍生物,承载大量生长因子,抗原物质少,制备容易,具有高效丝裂原效应,可能成为用于骨组织工程中生长因子的重要来源。
本研究目的旨在:1、探讨血液成分和骨组织之间的分子信号联络:GPR48基因失活小鼠在胚胎红系造血和骨发育调节中的共同分子信号通路;2、将血邪辶呀庖鹤魑ひ蜃釉靥?用于骨组织工程学方法,评价其在体内外对骨再生的影响。
材料和方法
1胚胎红系造血和骨发育的共同分子信号通路研究
1.1 GPR48基因靶向灭活小鼠品系的建立、基因型的确定及表达
利用基因分泌捕获方法建立GPR48基因靶向灭活的小鼠品系(129×C57BL/6),将GPR48杂合体雌雄小鼠交配饲养繁殖获得纯合突变小鼠子体,取孕中期12.5-14.5天(E12.5-E14.5)的杂合体雌鼠剖离胚胎做相关研究。
提取胎鼠尾DNA用PCR方法鉴定胚胎的基因型,区分GPR48野生型(WT,+/+)、杂合突变型(HE,+/-)和纯合突变型(HO,-/-)小鼠。用LacZ组织特殊染色观察GPR48在E14.5 WT、HO小鼠骨骼和肝脏的表达差异。
1.2体征观察
观察E12.5-E14.5胎鼠胚体和肝脏的体态大小、颜色、活动等一般情况。
1.3外周血红细胞及血红蛋白分析
制E12.5-E14.5外周血涂片,Wright-Giemsa染色,光镜下观察红细胞形态变化。对E13.5(WT 8只,HO 7只)和E14.5(WT 5只,HO 4只)血涂片,每只取3张血片,每张血片随取3个高倍视野,高倍镜(×400)计数单个视野的有核红细胞、无核红细胞及红细胞总数,计算有核红细胞在红细胞总数中所占比例,比较统计学差异;Real-time PCR检测E13.5 WT和HO各6例胎鼠外周血中βh1和β血红蛋白链的mRNA相对表达水平,比较统计学差异。
1.4 RT-PCR和Western-blotting对胎肝和骨骼ATF4表达分析
提取E13.5 WT和HO小鼠胎肝和肋骨笼的RNA和总蛋白,分别用RT-PCR和Western-blotting检测两种小鼠胎肝和骨骼中ATF4在mRNA和蛋白水平的表达差异。
1.5组织学及免疫组化增殖分析
取E13.5胎鼠的肝脏,常规10%中性福尔马林固定,乙醇逐级脱水,二甲苯透明,石蜡包埋,切取5μm切片,HE染色,光镜观察。按照相应的操作说明作胎肝(E13.5)和骨组织(E14.5股骨髁)的免疫组化分析,PCNA试剂盒用于增殖分析,ABC法用于ATF4的免疫组化分析,用苏木素复染。
1.6统计学处理
数据用均数±标准差表示,用SPSS11.5软件包做统计学分析,采用两个独立样本的非参数检验方法进行比较,P＜0.05为统计学有显著性差异。
2用组织工程学方法评价PL的生物学效应
2.1血小板裂解液对大鼠骨髓间充质干细胞生长和成骨分化的影响
2.1.1 BMSCs的分离、培养传代和表型鉴定
成年健康清洁级Wistar大鼠8只,体重250-300g,雌雄不限。采用全骨髓分离培养法培养扩增骨髓细胞,隔日半量换液,按1:3比例传代。倒置相差显微镜观察细胞生长。取第5代细胞,进行流式细胞仪检测细胞BMSCs表面特征抗原CD45/CD90/CD29的表达。
2.12 PL制备及生长因子含量测定
取16只大鼠,采用3次离心结合反复冻融法制备PL,0.22μm无菌滤膜过滤。取6只大鼠PL样品,按照ELISA试剂盒操作说明操作,测取PL中PDGF、TGF-β1、IGF-1和VEGF含量。
2.13 PL对细胞生长能力的影响
配制含PL终浓度为5%和1%(体积分数)两种不同的条件培养基。取第5代细胞,分A1(基础培养基中含5%PL)、B1(基础培养基中含1%PL)和C1(基础培养基)三组培养,用CASY细胞分析仪测定平均活细胞数,连续培养9天,每组每天测3孔,绘制细胞生长曲线,统计学分析不同浓度PL条件下细胞生长趋势和第9天时各组活细胞数的差异。
2.14 PL对BMSCs成骨诱导分化的干预效应
2.1.4.1建立成骨诱导体系取第4代细胞,以含有0.1μmol/L地塞米松、50μg/ml抗坏血酸、10mmol/Lβ-磷酸甘油钠及10%胎牛血清的H-DMEM为基础诱导液,配制含PL终浓度为5%和1%(体积分数)两种不同的条件诱导培养基,分别用于A2、B2组细胞的成骨诱导,C2组为不含PL的基础诱导液作为对照。连续培养20天,倒置相差显微镜下连续动态观察细胞形态变化和生长状况。
2.1.4.2不同PL诱导分化条件下BMSCs的ALP(碱性磷酸酶)活性和矿化形成
诱导7天时,各组细胞作ALP染色,观察胞浆内颗粒情况;诱导2、8和12天时,测取各组3个样本的ALP和TP(总蛋白)含量,以ALP/TP比值作为ALP活性定量指标。
诱导20天时,对细胞行Von Kossa染色和Alizarin red染色,观察细胞外基质矿化形成。对Alizarin red染色,每组取3例,每例随取5个视野,用图像分析软件计数单个视野中(×200)矿化结节数量和面积,比较各组差异。
2.2 PL/ADBG/CG与BMSCs的生物相容性研究
2.2.1同种异体脱钙骨颗粒(ADBG)的制备
大鼠10只(同上述),取四肢骨(包括干骺端),经深冻、超声清洗、粉碎后筛取直径300-500μm大小的骨粒,于超声条件下0.6N盐酸洗脱、冻干辐照。
2.2.2Ⅰ型胶原(CG)与ADBG的双相沉降复合
用0.1M乙酸作溶剂将CG配制成质量体积分数为0.25%的胶原溶液,辐照灭菌,4℃条件下将ADBG按质量体积分数为6%的比例加入CG溶液中,搅匀形成混悬液,加1N NaOH将混悬液的PH值调整到7.0左右,这时发生CG和ADBG发生共同沉降粘结,形成凝胶海绵样复合物,CG/ADBG的质量分数约为100mg/1g,取出后用无菌滤纸吸干,-20℃储存备用。
2.2.3 PL/ADBG/CG制备、复合培养
按5mL PL与5g ADBG/CG比例将两者在4℃无菌条件下浸泡24小时,大约60%PL被吸附于CG/ADBG。取PL/ADBG/CG复合物约100mg置于24孔培养板底部,与第4代BMSCs复合培养7d,倒置显微镜、电镜(3d)观察细胞形态及粘附生长状况。
2.3体内移植评价PL的生物学效应
2.3.1股骨髁缺损造模及材料移植
取大鼠(同上述)30只,分A、B、C三组,每组10只/20侧。无菌条件下制取大鼠双侧股骨髁内60-70%的腔隙性缺损。A、B和C组分别填塞植入等量(大约500mg/缺损)PL/ADBG/CG、ADBG/CG和CG。
2.3.2术后缺损修复效果评价指标
2.3.2.1 X线及骨密度计量分析
术后1-4周时,摄X光片作放射线检查了解新骨生长和缺损修复情况;用Image-Pro Plus 5.0图像分析软件测取2、4周缺损修复区单位面积的灰度值,每组测8个股骨髁,以代替骨密度评价缺损区骨修复状况。
2.3.2.2组织学观察及形态计量学分析
术后4周,每组随取3只动物,截取双侧股骨髁,经固定、脱钙、脱水、等常规病理操作方法,制作厚度5μm石蜡切片。常规HE染色,光镜观察。每组随取3张切片,每张切片随选5个视野(×200倍),用Image-Pro Plus 5.0图像分析软件测取缺损修复区单个视野下成骨区面积(以总像素数表示),比较修复效果。
2.3.2.3抗压生物力学测试
A、B、C每组随取实验动物3只,截取股骨髁,另取6具同窝大鼠正常股骨髁做对照D组。将股骨髁平置于载物台,压力装置以5mm/min的加载速度施压,电脑记录股骨髁位移达2mm时的破断载荷,并计算破断强度。
2.3.2.4三色流式细胞术测定外周血T-淋巴细胞亚群
4周处死动物前,A、B和C组各随选3只动物,另取三只同窝正常大鼠作为对照,麻醉后自心内采血2mL,利用三色流式CD4-FITC,CD3-PE和CD8-TC,检测各亚群百分比及CD4/CD8比值。
2.4统计学处理
用SPSS11.5软件包做统计学分析,数据用均数±标准差表示,采用析因分析交互效应,对于单因素效应采用One-way ANOVA,LSD法用于组间多重比较,P＜0.05为统计学有显著性差异。
结果
1 Gpr48-cAMP-CREB-ATF4分子信号转导共同调节胚胎红系造血和骨发育
1.1 GPR48基因的靶向灭活及其在胚胎期肝脏和骨组织表达
在小鼠Gpr48基因序列的内含子1区中随机插入了一个较大的分泌型捕获载体(11.9kb)。这个载体插入引起GPR48基因在小鼠基因组中表达缺失,PCR结果所显示的条带为750bp,而不出现GPR48正常基因条带(450bp)。LacZ组织学染色显示在胚胎E13.5肝脏和骨组织内均有GPR48表达,被染成蓝色,骨骼染色呈强阳性。
1.2 GPR48-/-小鼠发育迟滞和红系造血表型
大体观察发现与WT相比,E12.5-E14.5 HO胎鼠身体发育低下,体型、肝脏均明显减小,四肢骨骼发育短小,而且胎体和肝脏的颜色较苍白,皮下血管细微不发达。外周血涂片显示HO有核红细胞相对数明显升高,高倍镜下计数结果显示,E13.5天时HO和WT胎鼠的的有核红细胞在总红细胞数中所占的比例分别为(79.91±3.57)%和(30.00±2.17)%,HO比WT提高了约2.7倍水平,两者间比较,z=-3.24,P＜0.01,差异显著有意义;E14.5天时HO和WT分别为(54.99±1.93)%和(27.15±1.88)%,两者间比较,z=-2.45,P＜0.02,差异显著有意义。血红蛋白链Real-Time PCR分析结果:βh1链相对含量(WT为47.17±13.30,HO为113.50±23.44),β链相对含量(WT为136.50±20.42,HO为62.8±16.13)。与WT相比,HO胚胎血红蛋白βh1链mRNA相对量升高(z=-2.882,P＜0.01,n=6),差异有显著性意义;而成熟血红蛋白β的mRNA相对表达量则下调(z=-2.882,P＜0.01,n=6),差异有显著性意义。
1.3 ATF4在胎肝和骨中的表达分析
胎肝和骨RT-PCR结果显示,HO胎鼠ATF4在约400bp处出现的条带亮度明显弱于WT对照;Western-blotting结果同样显示在约40kD处,HO出现的条带亮度明显弱于WT对照。免疫组化分析显示,ATF在HO胎鼠肝脏和骨组织中的表达呈现弱阳性,显著低于WT中ATF4的表达。
1.4组织学及增殖分析
肝脏HE染色结果显示,纯合体与野生型相比,肝细胞形态类似,但定向红系祖细胞和红细胞岛明显减少。PCNA增殖分析结果显示HO胎肝(E13.5)和骨组织(E14.5股骨髁)呈现弱阳性染色,处于增殖期的细胞明显少于WT,E14.5HO股骨皮质骨厚度也明显减少。
2用组织工程学方法评价PL的生物学效应
2.1 PL对大鼠体外BMSCs生长和成骨分化的影响
2.1.1 BMSCs的分离、培养、传代和表型鉴定
BMSCs经分离培养传至第5代时即纯化为单一纺锤状,规则平行排列,类似于成纤维细胞形态。流式细胞仪分析,这些细胞群落表达的BMSCs特征表面抗原高度均一,呈现CD45~-CD90~+CD29~+,符合BMSCs的表型特征。
2.1.2 PL中生长因子含量
经ELISA定量分析,PL中PDGF、TGF-β1、IGF-1和VEGF浓度分别为405±59.58、140±26.65、85.83±16.86和82.5±11.73(pg/mL)。
2.1.3不同浓度PL对BMSCs增殖的影响结果
生长曲线显示自第3天开始细胞生长迅速,随着PL浓度的提高和时间延长,各观察点活细胞数呈现递升趋势;A1组细胞增殖较快,在第3天达到对数生长期,比C1组提前约1天。析因分析结果显示,不同浓度的PL对BMSCs的增殖差异有显著性意义(F=320.33,P＜0.01),培养时间对BMSCs的增殖差异也有显著性意义(F=406.04,P＜0.01),PL浓度和培养天数之间有交互效应(F=36.25,P＜0.01),说明随着PL浓度的升高和培养时间的延长细胞增殖呈上升趋势。进一步用TUkey HSD法做差异性检验的多重比较,结果显示A1组对细胞增殖的影响高于B1(P＜0.01)、C1组(P＜0.01),B1组对细胞增殖的影响高于C1组(P＜0.01),差异有显著性意义。
第9天时各组活细胞数分别为A1(988436±15720.42)个、B1(616666.67±12423.1)个和C1(345000±21931.71)个,单因素方差分析结果显示各组间差异有显著性意义(F=1064.11,P＜0.01);进一步组间多重LSD比较显示,A1组活细胞数高于B1(P＜0.01)组和C1组(P＜0.01),B1组高于C1组(P＜0.01),差异有显著性意义,其中细胞数达到C1组的近3倍水平。
2.1.4不同PL条件下对BMSCs成骨诱导分化的影响
2.1.4.1不同诱导条件下对BMSCs ALP活性的影响
不同PL条件成骨诱导分化下,C2组细胞长突起逐渐变短,胞体变宽、变大,7d时胞浆内出现较多ALP染色阳性的粗大颗粒。B2组与C2组细胞的形态改变相类似,但程度不如A2组明显,而且细胞增殖较C2组快。A2组细胞生长较B2、C2迅速,细胞形态改变不如B2、C2两组显著,胞浆颗粒不发达,细胞处于迅速增殖状态。20天时,B2和C2组细胞外有大量矿物沉积;A2组细胞仍呈密集生长,排列杂乱,细胞外基质中沉积物明显少于B2、C2两组。
ALP活性定量分析中,第2、8、12天A2、B2、C2组ALP活性析因分析显示,不同诱导条件下ALP活性的差异有显著性意义(F=45.698,P＜0.01);进一步组间LSD多重比较显示,P_(A2-B2)＜0.01,P_(A2-C2)＜0.01,差异有显著性意义,P_(B2-C2)＞0.05。不同培养天数间差异有显著性意义(F=121,P＜0.01),不同的PL浓度与天数对ALP活性的影响存在显著性交互效应(F=6.86,P＜0.01),以C2组在第12天时的ALP活性最高。
2.1.4.2不同诱导条件下对BMSCs基质矿化的影响
B2、C2组细胞在20天时在胞外基质中出现大量沉积物,而A2组20天时细胞仍密集生长,胞外基质中沉积物较少。Von Kossa染色和Alizarin red染色显示A2两组矿化沉积明显少于B2、C2组。Alizarin red染色单个视野(×200倍图像)中各组矿化结节计数A2、B2和C2组分别为7.67±1.10、14.0±2.23和14.97±1.88(个);组间方差分析显示F=14.593,P＜0.01,差异有显著性意义;进一步组间LSD两两比较,A2组矿化结节数显著低于B2(P＜0.01)和C2(P＜0.01),差异有显著性意义,而B2与C2间无显著性差异(P＞0.05)。
A2、B2和C2矿化结节的面积分别为161778.73±44550.80、337349.67±56083.24和415921.73±71725.39(像素),组间方差分析显示F=14.831,P＜0.01;进一步组间LSD两两比较,A2组钙结节面积显著低于B2(P＜0.01)和C2(P＜0.01),差异有显著性意义,而B2与C2间无显著性差异(P＞0.05)。
2.2 PL/BDG/CG与BMSCs体外共育的生物相容性
共育3、7d,倒置相差显微镜显示BMSCs细胞形态无明显异常,材料边缘及周围可见大量BMSCs生长。扫描电镜显示细胞呈长梭形,有多个长突起,紧密贴附于材料表面,融合呈网状连接,有足突状突起向材料内部深入生长。
2.3 PL/ADBG/CG体内移植对股骨髁缺损修复重建效果
2.3.1放射学及骨密度计量学分析
A组,1周时缺损修复区可见少量稀疏絮状阻射影;2周时絮状阻射影密度较前增加;3周时阻射影质地渐趋均匀、致密;4周时缺损中心区阻射影密度接近正常骨质,缺损基本修复。B组,1周时缺损中心区为大量透亮区;2周时阻射影密度较前增加;3周时仍有较多不规则的透亮区;4周时阻射影密度趋于致密,仍有少量不规则的透亮区。C组,1周时缺损仍为明显的透亮区;2周时,A组缺损区有少量模糊阻射影;3周时中心区仍以大量透光区为主;4周时缺损未见修复迹象。
灰度(代骨密度)计量分析显示,2周时,组间方差分析F=25.08,P＜0.01,各组灰度值间差异有显著性。进一步组间LSD多重比较,A组单位面积的灰度值高于B组(P＜0.01)和C组(P＜0.01),B组高于C组(P＜0.05),差异有显著性意义。4周时方差分析F=29.08,P＜0.01,各组灰度值间差异有显著性。进一步组间多重比较,A组单位面积的灰度值高于B组(P＜0.01)和C组(P＜0.01),B组高于C组(P＜0.05),差异有统计学意义。
2.3.2组织学及组织计量学分析
4周时,A组修复区可见已成活骨片,有较多成骨细胞和新生类骨质长入,也可见正在被吸收的骨片,周围包裹大量纤维组织,伴少量炎细胞浸润;正常骨端向缺损区有大量新生骨延伸和多处软骨内成骨现象,成骨和破骨活动均较B和C组活跃。B组仍有较多死骨存在,可见崩解的碎片和正在被吸收的骨颗粒,有少量骨颗粒成活,但成骨和破骨现象不如A组活跃。C组仍填充以大量纤维组织,周围多处正常骨壁成骨不活跃。
经骨组织形态计量学分析,对单个视野(×200倍)中其新生骨软骨和成活的移植骨片总面积(像素值)作方差分析,F=122.398,P＜0.01,差异有显著性意义;进一步作组间两两多重比较,结果显示A组成骨面积高于B组(P＜0.01)和C组(P＜0.01),B组高于C组(P＜0.01),差异有有显著性意义。
2.3.3抗压生物力学评价
各组标本经抗压生物力学测试,对破断载荷和破断强度做方差分析,F=64.469,P＜0.01,差异有显著性意义;进一步作组间两两多重比较,结果显示A组破断载荷和破断能量均高于B(P＜0.01)和C组(P＜0.01),但低于正常对照D组(P＜0.01),B组显著高于C组(P＜0.01),C组最低,差异有显著学意义。
2.3.4三色流式细胞术T细胞亚群测定
三色流式细胞术分析结果见表2.3.3,经方差分析,CD3~+CD4~+CD8~-、CD3~+CD8~+CD4~-、CD4/CD8在各组间均无显著性差异(P＞0.05)。
结论
血液成分(包括红细胞和血小板)与骨组织的发育、再生是存在内在联系的,血小板裂解液可替代多种生长因子应用于骨组织工程方法。
1.GPR48-cAMP-CREB-ATF4分子信号转导是红系造血和骨发育共同的转导信号控制通路之一,同时调控小鼠胚胎期红细胞的成熟和骨发育,构成连通血液系统和骨骼组织的分子信号桥梁。GPR48的灭活同时造成红系造血和骨发育缺陷,这对解释临床上某些不明原因的疾病具有潜在的参考价值。
2.GPR48信号通路通过调节细胞增殖影响骨和红细胞发育。
3.PL由血小板衍生而来,是多种生长因子的承载体系,可参与骨形成调控。PL以剂量依赖方式,对大鼠BMSCs发挥丝裂原作用,促进细胞增殖;而对BMSCs成骨诱导分化过程中的ALP活性和矿化形成呈现剂量依赖性的抑制作用。PL对BMSCs增殖和分化的不一致效应可能对早期骨修复有利。
4.PL可作为生长因子用于骨组织工程学方法,所构建的重组合异体脱钙颗粒骨PL/ADBG/CG与BMSCs有良好的生物相容性,复合移植后可促进骨缺损的早期修复过程。
5.PL对骨重建有促进作用,可能与多因子协同作用下激发骨吸收-骨形成偶联机制有关。
Background and purposes of the research
Bone tissue engineering refers to a process to mimic physiological bone regeneration. The connection of molecular signal mechanism exists between blood system and skeleton, on which performing profound research helps to the further development in bone tissue engineering. G protein coupled receptors (GPCRs) represent the first switch not only to receive signals from outside, but also to transduct molecular signals to intracellular transcript factors which function to regulate the biological activities of various cells. G protein coupled receptor 48 (GPR48) gene knock-out fetal mice showed both anemia appearance and marked development retardation of skeleton, this phenotype suggested that some molecular connection existed between hematopoiesis and skeleton, but we haven't made it clear. Therefore, it is helpful to obtain profound acknowledge about bone regeneration under the research on the molecular connection between hematopoiesis and skeleton development through GPR48 signal pathway.
In recent years, varied components in blood, especially platelet, have attracted more and more attention due to the important role they played in bone regeneration. Blood platelets function to regulate the proliferation and differentiation of varied cells including osteoblast and osteoclast by way of releasing various growth factors when activated by coagulate mechanism. The maintenance and activation of normal platelets' functions depends on the regulation by many signal pathways via GPCRs. Therefore blood platelet forms the bridge to connect blood system with bone repair through GPCRs pathway. Most resently, platelet lysate (PL), which has been reported to be derived from platelet and loaded with large amount of growth factors and less antigen, functions as a highly effective mitogen, all of which implies that PL might be a potential resource of growth factor substitutes applied into bone tissue engineering.
The purpose of this reaseach aims: 1) To explore the molecular connection between blood components and skeleton tissue: the shared molecular signal pathway in erythropoiesis and skeleton development for GPR48 gene-traped fetal mice; 2) To evaluate the effects of PL as a resorce of growth factors on bone regeneration by way of bone tissue engineering strategies both in vitro and in vivo.
Material and Methods
1 Study on the shared molecular signal pathway functions both in fetal erythropoiesis and skeleton development
1.1 Generation of GPR48 knocked out mice, determination of genotype and expression pattern
Gpr48 knocked out mice of (129×C57BL/6) were generated via secretory-trap approach. Homozygous were obscured by intercrossing heterozygous mice. E12.5～14.5 fetal mice isolated from HE female pregnant mice were selected for this research program.
Genotype of mice (wild-type +/+, heterozygous +/-, and homozygous -/-) was determined by PCR to distinguish the type of GPR48 genomic DNA extracted from the tails of fetal mice. LacZ histochemistry assay was carried out to detect the discrepancy of GPR48 expression in skeleton and liver between GPR48 wild-type and homozygous fetal mice.
1.2 Grossic phenotype
To observe the gross characteristics including size, color both for embryos and liver, the size of liver and action status of fetal mice obscured from E12.5～14.5 embryos.
1.3 Analysis of erythrocytes and hemoglobin chains in peripheral blood
Blood smears were prepared by wedge technique following Wright-Giemsa staining protocol for E12.5～14.5 embryos. For E13.5 fetal mice (8 WT and 7 HO) and E14.5(5WT, 4HO), 3 blood smear slides were randomly selected from each embryo in WT or HO, and 3 high power views of each slide were randomly chosen to count the number of nucleated erythrocytes and enucleated erythrocytes, and calculated the relative level of nucleated erythrocyte in total number of erythrocytes for comparisons.
Real-time PCR was performed to detect the mRNA level ofβh1 andβhemoglobin chains in blood of E13.5 WT (6 embryos) and HO (6 embryos).
1.4 ATF4 expression in fetal liver and skeleton
The total RNA and protein extracted from liver and bone (lib cage) of both E13.5 wild-type and homozygous embryos were detected by RT-PCR and Western-blotting approaches respectively to assay ATF4 expression in liver and skeleton (lib cage) at both of mRNA and protein level.
1.5 Histology and Immunohistochemistry (IHC) assay
Fetal liver (E13.5) dissected from mice were fixed in 10% formalin and embedded in paraffin according to the standard techniques. Five micrometer-thick sections were stained with hematoxylin and eosin (H&E). For proliferation assays, the sections of both the liver (E13.5) and femur (E14.5) were stained with anti-PCNA using PCNA kit and then counterstained with hematoxylin. For ATF4 expression in liver and bone, the following antibodies were used: rabbit polyclonal anti-ATF4 (C-20) (1:100), hematoxylin was carried out as counterstain.
1.6 Statistical analysis
Data were presented as mean±standard deviation and analyzed by 2-independed samples non-parameteric test with SPSS 11.5 software. For all analyses, P < 0.05 was considered statistically significant.
2 Evaluation of the biological effects of PL via the strategies of tissue engineering
2.1 Effects of PL on the proliferation and osteogenic differentiation of BMSCs.
2.1.1 Isolation, expansion and phenotypic analysis of BMSCs
Total 8 adult healthy Wistar rats with weight of 250-300g of clean grade were used in this section, whole bone marrow was cultured and expanded by changing medium in half every other day and passaged at a ratio of 1:3. The cell growth was observed by inverted microscope. The 5th passages were prepared to determine the expression of cell surface antigens CD45/CD90/CD29 which function as the specific marker of BMSCs by flow cytometry.
2.1.2 Preparation of PL and ELISA assay for the determination of growth factors
PL was obtained through three times of centrifugations and repeated freeze-thaw for the venous blood aspirated from 16 rats and filtrated through 0.22μm sterilized filter membrane. ELISA assay was conducted following the manufacture's protocol to determine the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL samples from 6 rats.
2.1.3 Effects of PL on cell growth
PL at final concentration of 1% and 5% in basic medium (V/V) was prepared as conditioned medium. BMSCs of the 5th passages were classified into three groups of A1 (5% PL in basic medium) , Bl(l% PL in basic medium), and C1(no presence of PL in basic medium as controls) to expand BMSCs respectively for a series of 9 days so as to draw the cell growing curves through the assay by CASY cell analyzer. The living cell number of all groups was compared at 9d.
2.1.4 Effects of PL on osteogenic differentiation of BMSCs
2.1.4.1 The establishment of osteogenic differentiation system
The cells at fourth passage were performed for osteogenic differentiation under induction environment in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as controls). The morphological changes of cells were dynamically observed with inverted phase contrast microscope.
2.1.4.2 ALP (Alkaline phosphatase) activity and mineral formation of BMSCs under inductive differentiation with different PL concentration
ALP staining (7d) and ALP/TP (2, 8,12d) of the cells were detected to evaluate ALP activity for 3 samples of each group. At 20d, Von Kossa and Alizarin red staining were both carried out to observe extracellular mineral formation. For Alizarin red staining, 3 cases of each group were used to examine the mineral formation in extracellular martrix, randomly chose 3 views (×200) to obtain the number and area of mineralized nudes under microscope for each case with image analyis software.
2.2 Study on the biocompatibility between PL/ADBG/CG and BMSCs
2.2.1 Preparation of allogeneic decalcified bone granules (ADBG)
10 Wistar rats were used to prepare ADBG, the limbs bone were processed through deep freeze, supersonic clean, grinding to obtain granules with diameter range of 300-500μm. The granules were further processed through 0. 6N HCl washing, lyophilization and radiation.
2.2.2 The combination of Collgen I (CG) with ADBG via dural-phase precipitation
The CG resolution at the concentration of 0.25% (W/V) was prepared using 0.1M acetic acid as basic solvent and sterilized by radiation. ADBG was added into this CG resolution according to W/V fraction of 6% and mixed well to obtain a suspension, to which IN NaOH was added to adjust the PH value to 7.0 approximately, then CG and ADBG precipitated and adhered together to form a gel-like composite CG/ADBG (100mg/1g), which then was dried on the filter paper and stored at -20℃.
2.2.3 Preparation of PL/ADBG/CG and co-culture with BMSCs
PL/ADBG/CG was obtained by immersing 5g ADBG/CG with 5mL PL under aseptic condition at 4℃. BMSCs of fifth passage were coincubated with PL/ADBG/CG for 7d to observe the biocompatibility and adhensive growth with inverted microscope, and scanning electron microscope was conducted for the composite at 3d.
2.3 Effects of PL/ADBG/CG transplantation on reconstruction of femoral condylar defect in vivo
2.3.1 Establishment of femoral condylar defect model and graft transplantation
30 Wistar rats as above description were devided into 3 groups (A, B, C) with 10 rats/20 condyles in each group. The bilateral femoral condylar defects (with 60-70% bone loss) were established for these animals and transplanted with 500mg of A(PL/ADBG/ CG), B(ADBG/CG) or C(CG) filled into defect respectively.
2.3.2 Evaluation on bone defect repair after operation
2.3.2.1 X-ray and bone density assay
After operation, X-ray was performed to observe the new bone formation and repairing status. Image-Pro Plus 5.0 software was used to measure the gray level per unit area in repairing field of 8 femoral condyls in each group at postoprerative 2 and 4w, which represented the bone density for the evaluation on bone defect repair.
2.3.2.2 Histology and morphological quantitative analyses
At 4 weeks, the femoral condyles of 3 rats in each group were cut off and processed by fixation, decalcified, dehydration, and other procedures to make paraffin sections with thickness of 5μrn. After regular HE staining, microscope was conducted to observe the bone formation, 3 sections were randomly selected and 5 views in each section under microscope (×200) were randomly chosen to measure the new bone area (pixels) per view (×200) with Image-Pro Plus 5.0 software.
2.3.2.3 Biomechanical assay of anti-pressure
At 4 weeks, 6 condyles from 3 rats in each group were used for mechanical test together with 6 normal condyles of littermates as group D. Condyles were horizontally placed on the object stage, and pressed at the loading speed of 5mm/min. The destructive loading was recorded when displacement attained to 2mm and calculated the destructive intensity.
2.3.2.4 Determination of T lymphocyte subsets in peripheral blood
Before killing rats at 4w, 3 rats were randomly choosed from A、B and C ,and another 3 rats littermates were performed as controls. Three-color flow cytometry was conducted by using CD4-FITC, CD3-PE and CD8-TC to detect percentage of T lymphocyte subsets as well as the ratio of CD4/CD8.
2.4 Statistical analysis
Data were presented as mean±standard deviation and analyzed by Factorial analysis for interaction effects, and One-way ANOVA was performed for one-factor effect. LSD was conducted for related multiple comparisons. For all analyses, P<0.05 was considered statistically significant.
Results
1 GPR48-CAMP-CREB-ATF4 signal transduction referred to the shared molecular signal pathway to regulate the development of both erythropoiesis and skeleton at embryonic stage
1.1 Targeted inactivation of GPR48 and expression pattern in fetal liver and skeleton
The murine GPR 48 genomics was disrupted by randomly inserting a large secretory trap vector (11.98kb) to the intron 1. The insertion caused deletion of GPR48 in mouse genome, which was confirmed by PCR analysis which showed that GPR48-/- null mice didn't present the band of normal GPR48 gene at 450bp. LacZ staining showed that GRP48 was expressed both in E13.5 fetal liver and skeleton in blue color.
1.2 Growth retardation and erythropoiesis phenotype
The gross appearance of GPR48-/- null fetal mice from E12.5 to E14.5 showed growth retardation both in body size and liver size, limbs were shorter and both body and liver were markedly paler when compared with wild-type controls.
The peripheral blood smear for E13.5～E14.5 indicated that HO had more nucleated erythrocytes in blood. E13.5 blood smear assay displayed the proportion of nucleated erythrocyte in total erythrocytes of GPR48-/- null fetal mice elevated about 2.7 folds of normal litters, which with significant difference between these two groups (HO 79.91±3.57, WT 30. 00±2. 17; z=-3.24, P<0.01). For E14.5 mice, there also was markedly difference between HO and WT (HO 54.99±1.93, WT 27.15±1.88; z=-2.45, P<0.02).
Real-time PCR showed that relative mRNA level ofβh1 in E13.5 homozygous blood was significantly increased than that in normal littermates (z=-2.882, P<0.01). Otherwise, the relative mRNA level ofβin homozygous blood was markedly reduced than that in wild-type littermates (z=-2.882, P<0.01).
1.3 ATF4 expression in GPR48-/- fetal liver and skeleton
In RT-PCR, ATF4 expression in E13.5 GPR48-/- fetal liver and skeleton (lib cage) showed that HO presented a significantly weaker band at 400bp than WT. In Western-blotting, HO also presented a markedly weaker band at 40kD than WT. Immunohistochemistry assay indicated that ATF4 expression in both HO liver (E13.5) and skeleton (E14.5 femoral condyle) was weakerly in positive staining than normal controls.
1.4 Histology and proliferation assay
Histological examination and hematoxylin and eosin (H&E) staining showed that the gross morphologic features of homozygous fetal livers were normal in cellular architecture. However, fewer erythroid precursor or definitive progenitors and erythroid foci were visible in the homozygous fetal livers from E13.5 embryos compared with that in wild-type.
Immunohistochemical assays (IHC) of PCNA for proliferation assay indicated that the thickness of femoral cortex in E14.5 HO was significantly thinner than that in WT control. The strength of proliferating stain was markedly weaker in the fetal livers and femoral condyles of homozygous Gpr48 null mice compared with their control littermates.
2 Evaluation of the biological effects of PL via the strategies of tissue engineering
2.1 Effects of PL on proliferation and osteogenic differentiation of BMSCs
2.1.1 Isolation, expansion and phenotypic analysis of BMSCs
Under inverted microscope, cells at 5th passage presented the similar morphology of uniform long-spindle shape as fibroblasts and in regular arrangement of parallel aggregation. The cells expressed CD45~-CD90~+CD29~+ antigens by assay of flow cytometry which conformed to the characterized cell surface antigens of BMSCs.
2.1.2 ELISA assay of the concentration of growth factors in PL
ELISA assay presented that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL attained to 405±59.6, 140±26.65, 85.8±16.86, and 82.5±11.7 (pg/ml) respectively.
2.1.3 Effects of PL on cell proliferation
Under different conditioned medium, BMSCs in A1, Bl and C1 displayed nearly the same configuration with long shuttle-shape as fibroblasts. The growth curve displayed that cells grew faster from 3d and the living cells number at every timepoint presented ascending trend along with the prolongation of culture time. Cells in A1 grew fastest within these 3 groups from 3d into logarithmic growth phase which was 1 day earlier than that in Cl. The results of Factional analysis showed that different PL concentration had significant difference in effects on the cell proliferation (F=320.33, P<0.01), while different culture time also markedly affect the cell proliferation (F=406.04, P<0.01), and there was interaction effect between different PL concentration and cultural time (F=36.25, P<0.01) which certificated that cell proliferation presented ascending trend along with the prolongation of culture time in different PL concentration. Further LSD assay of the multiple comparations presented that cell proliferation of group A1 was higher that that of B1 (P <0.01) and C1(P <0.01), and that B1 was higher than C1(P <0.01), these differences were significantly different.
At 9d, the living cell number in A1, B1 and C1 was 988436±15720.42, 616666.67±12423.1, and 345000±21931.71 respectively, One-way ANOVA showed there was significant difference within these groups (F=1064.11, P<0.01). Further multiple comparisons with LSD showed that the living cell number in A1 was significantly higher compared with that in B1 (P <0.01) or C1(P <0.01), and living cell number in B1 was higher than that in C1(P <0.01), there differences were significant within them. The living cell number in A1 reached about 3 folds of that in C1(P<0.01) at 9d.
2.1.4 Effects of PL on the osteogenic differentiation of BMSCs
For differentiation analysis, morphological observation displayed BMSCs in group A2 showed slower shape-changes but higher proliferation than that in group B2 or C2. Cells in B2 showed higher proliveration than that in C2 but lower that that in A2. At 7d, the cells in group A2 showed smaller amount of granules with ALP positive staining in cytoplasm when compared with B2 or C2. Moreover, at the day of 20, the cells in group A2 still displayed much higher dense growth.
At the day of 2, 8, and 12, Factorial assay of ALP activity showed there was marked diffence within these groups (F=45.698, P<0.01). Further multiple comparisons showed marked reduce of ALP activity in A2 compared with that in B2 (P <0.01) or C2 (P <0.01) and B2 displayed no significant decrease compared with C2 (P >0.05). Different days showed significant difference to affect the ALP activity (F=121, P<0.01). There was interaction effect between different days and different PL concentration (F=6.86, P<0.01) , the highest value of ALP activity occurred at 12d.
At 20d, both Von Kossa and Alizarin red staining showed there were large amount of mineral deposits occurred in the excellular martrix of B2 and C2, but smaller amount in A2 compare with B2 and C2.
In Alizarin red staining, the mineral nodes under the single view (×200) attained to A2 (7.67±1.10), B2 (14.0±2.23), C2 (14.97±1.88) respectively. With One-way ANOVA assay, there was significant difference in the nodes number under the single view within these groups (F=14.593, P<0.01), further LSD asaay showed significantly smaller amount of mineral deposits produced in the excellular martrix of A2 compared with B2 (P <0.01) or C2 (P <0.01), while no marked difference between B2 and C2 (P>0.05). The area of mineral nodes under the single view (x200) attained to A2 (161778.73±44550.80), B2 (337349.67±56083.24), C2 (415921.73±71725.39) pixels respectively. With One-way ANOVA assay, there was significant difference in the area of nodes under the single view within these groups (F=14.831, P<0.01), further LSD asaay showed significantly larger area of mineral deposits produced in the excellular martrix of A2 compared with B2 (P <0.01) or C2 (P<0.01) , while no marked difference between B2 and C2 (P>0.05).
2.2 The biocompatibility of PL/ADBG/CG with BMSCs
After 3d and 7d in coculture, BMSCs grew normally around or adhered to PL/ADBG/CG with no significant shape change under inverted microscope. Under scanning microscope at 3d, living cells with long processes tightly adhered to the surface and grew toward the deep inner of the composite.
2.3 Effects of PL/ADBG/CG transplantation on reconstruction of femoral condylar defect in vivo
2.3.1 Radiology and quantitative assay of bone density
In group A, X-ray at 1w showed small amount of resistance projective image in repairing region occurred; at 2w, the density of resistance projective image was increased than before; at 3w, the image of resistance projective gradually tended dense and homogenous; at 4w, the image of bone density was close to normal bone nearby and the defect was nearly repaired. In group B, at 1w the center of defect region showed large amount of translucent region; at 2w, the image density was increased than before; at 3w, there was more irregular translucent region; at 4w, the defect center majored large amount of translucent region and no clues of defect repair.
These observations were conformed by quantitative assay of bone density which showed that there was dramatic difference within A, B, and C (2w: F=25.08, P<0.01; 4w: F=29.048, P<0.01). Further multiple comparisons presented that at either 2 or 4 weeks, the bone density in A presented significantly higher than that in B (P <0.01) or C (P <0.01), and B was also significantly higher than C (P <0.05) in bone density.
2.3.2 Histology and quantitative assay
Histology assay at 4 weeks displayed that there were more osteoblast and new osteoid growing into bone granules, also the phenomenon of absorption was easy to be seen in the dead bone which surrounded by fibrous tissues with small amount of inflammatory cells infiltration. Group A showed large amount of new bone forming from normal bone nearby expanded and more endochondral ossification could be observed. Moreover, there were more osteoblastic and osteoclastic activities in A than in B or C. There were more dead bone pieces in disintegration and absorption and small amount of new bone formation occurred in defect region of group B. There were still large amount of fibrous tissues to fill the defect field in group C, many sites of normal bone walls showed less active in ossification.
Quantitative assay displayed the newly reparative bone area within these 3 groups was dramatically different (F=122.398, P<0.01), and further LSD multiple comparisons showed that the newly formed bone area in A was significantly higher than that in B (P <0.01) or C (P <0.01), and B showed more bone formation than C (P
2.3.3 Anti-press biomechanical evaluation
Anti-press mechanical measures showed that there was significant difference within A, B, C, and D (F=64. 469, P<0. 01). Further multiple comparisons were conducted and the results showed that either destructive load or destructive energy was higher in A than that in B (P <0.01) or C (P <0.01), but markedly lower than in D (P <0.01) which referred as normal controls, while C presented the worst result with statistic significance (P <0.01).
2.3.4 T lymphocyte subsets determination
The T lymphocyte subsets of CD3~+CD4~+CD8~-, CD3~+CD8~+CD4~- and the ratio of CD4/CD8 showed no significant difference within these 4 groups by One-Way ANOVA assay (P>0.05).
CONCLUSION
Blood components incluling erythrocytes and platelets present the internal connection with skeleton tissues either in development or bone regeneration, and platelets functioning as a resorce of multiple factors may be applied to the strategies of bone tissue engineering.
1 GPR48-cAMP-CREB-ATF4 signal transduction, which is one of the shared molecular signal pathways to regulate the development of both erythrocyte and skeleton in fetal mice, forms the molecular bridge to connect blood system with skeleton tissue.
2 GPR48 signal pathway regulates the development of both erythrogenesis and skeleton via cell proliferation.
3 PL is derived from platelet and presents to be a kind of system carrying various growth factors to regulate bone formation. PL functions as a kind of mitogen to promote the proliferation of BMSCs in a dose-dependent manner, while inhibits both of ALP activity and mineral formation of BMSCs also in dose-dependent manner under osteogenic induction environment. But this non-coordination effects of PL on proliferation and differentiation implies that PL may function to promote the bone formation at earlier stage.
4 PL presents to be a nice substitute for growth factors and can be applied to the strategy of bone tissue engineering. PL/ADBG/CG, the recombinant decalcified allograft bone, which shows better compatibility with BMSCs, can promote bone defective repair at early stage.
5 PL promotes the bone remodeling in repairing the femoral condylar defects, which may be related with the activation of coupling mechanism between bone absorption and formation via the synergistic effects of various factors.
引文
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