普伐他汀对BMSCs复合FS移植治疗兔早期激素性股骨头坏死的干预研究
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摘要
目的：
双次注射内毒素加三次注射激素建立兔早期激素性股骨头坏死模型,探讨其可能的发病机制；构建可注射型FS包埋Brdu标记的BMSCs的工程化组织,验证其生物相容性以及体外成骨诱导分化的效果；观察普伐他汀在体外对BMSCs/FS成骨诱导分化及分泌VEGF, BMP-2的影响；观察普伐他汀对BMSCs/FS经髓芯减压移植治疗兔早期激素性股骨头坏死的干预效果,并探讨其作用机理。
方法：
第一部分：兔早期激素性股骨头坏死模型建立：取健康成年新西兰大白兔80只,雌雄不限,随机分成4组,每组各20只。A组：(对照组)与D组相同时间点注射等量生理盐水。B组：马血清加三次注射激素组；C组：单次注射内毒素加三次注射激素组；D组：双次注射内毒素加三次注射激素组。注射后观察动物一般情况。各组于用药后第4、6、8、10、12周行血清胆固醇、甘油三酯,血液流变学、凝血功能,血栓调节蛋白含量等检测,并应用SPSS17.0版统计软件,两组数据间比较采用t检验,多组数据间比较则采用单因素方差分析,进行q检验,以P<0.01作为统计学具有显著性差异的标准对数据进行分析处理。于第6、8、10、12周处死兔子取双侧股骨头行大体标本、常规组织病理学切片及血管内皮生长因子免疫组化染色、股骨头微血管内血栓染色,于注射前及末次注射后4、8及12周,行X线片、MRI检查,观察股骨头影像学改变。
第二部分：构建可注射型FS包埋Brdu标记的BMSCs的工程化组织：取健康新西兰大白兔8只,雌雄不限,采用全骨髓培养法,经过换液及传代,贴壁生长细胞通过流式细胞仪检测CD34、CD44等表面标记物,证实为高纯度的BMSCs后；取P3代BMSCs,分为四组：A组(对照组)：BMSCs+成骨培养基培养；B组(BrdU组)BMSCs +BrdU+成骨培养基培养；C组(生物蛋白胶组)：BMSCs+生物蛋白胶+成骨培养基培养；D组(生物蛋白胶+BrdU组)：BMSCs+生物蛋白胶+BrdU+成骨培养基避光培养。成骨诱导1、2、4、7、14、21天后,分别行光镜下大体观察,生长动力学检测、台盼蓝染色、碱性磷酸酶Gomori钙钴法和Von-Kossa改良法染色鉴定向成骨方向分化的能力,检测碱性磷酸酶定量和游离钙定量检测(甲基百里香酚蓝比色法),采用SPSS17.0统计软件对数据进行统计分析。
第三部分：观察普伐他汀在体外对BMSCs/FS成骨诱导分化及分泌VEGF,BMP-2的影响：BrdU标记的P3代BMSCs/FS加入成骨培养基避光培养,设对照组(A组)：加入等量溶剂,实验组(B组)：加入普伐他汀(100umol/L)。细胞培养1、4、7、14天在倒置显微镜下观察细胞形态,进行生长动力学检测,14天碱性磷酸酶(alkaline phosphatase,ALP)活性测定,21天Von-Kossa改良法染色鉴定向成骨方向分化的能力,第21天提取纯化mRNA,逆转录合成cDNA,荧光标记后与全基因组寡核苷酸芯片杂交,扫描后筛选出差异表达基因。第14天采用Elisa法检测VEGF和BMP-2表达量。采用SPSS17.0统计软件对数据进行统计分析。
第四部分：观察普伐他汀对BMSCs/FS经髓芯减压移植治疗兔早期激素性股骨头坏死的干预效果：取造模4周后存活的成年新西兰白兔60只,雌雄不限,随机分为四组每组15只：A组：(对照组)不做任何处理；B组：(髓芯减压组)C臂X光透视下用骨穿针行股骨头髓芯减压；C组：(干细胞移植组)股骨头髓芯减压同时局部移植Brdu标记的BMSCs/FS, D组：(干细胞移植联合普伐他汀治疗组)在C组基础上次日起以普伐他汀灌胃。造模后4周、8周、12周及16周,抽取耳缘静脉血检测血脂。造模后8周、12周、16周分批处死兔子,截取双侧股骨头,分别进行大体观察、常规组织病理学切片光镜和电镜观察。8周、12周及16周行血管内皮生长因子、核心结合因子免疫组化染色,8周、12周及16周实时荧光定量检测VEGF和Cbfa1mRNA的表达。造模后8周C、D两组行Brdu标记干细胞示踪检测。造模后8周、12周及16周,行双侧股骨头1.5T核磁共振(MRI)检测观察坏死信号的改变。采用SPSS17.0统计软件对数据进行统计分析。
结果：
第一部分：A组正常,B、C、D组动物于末次注射后3周体重减轻,精神萎靡,活动及饮食减少,反应淡漠,分别有4、5、7只动物死亡,各期血清胆固醇、甘油三酯、低切变率下全血黏度、血浆黏度、红细胞聚集指数均明显升高(P＜0.05),以D组更为明显。自造模后10周始,两组间各指标比较均有统计学差异((P＜0.05)。注射前及注射后2周各组MRI显示正常,C、D组4周股骨头信号轻度减低,A、B组未见异常。C、D组8周股骨头有小部分坏死区域,B组信号轻度减低,12周后D组股骨头坏死区域进一步扩大。B、C、D组空骨陷窝率分别为10.4%±4.4%,19.7%±4.7,33.2%±5.6%。骨小梁面积百分比分别为64.4%±8.4%,53.2%±±5.7%,41.3%±5.1%；D组指标与B,C组比较,差异有显著统计学意义(P<0.01)。影像学及病理学研究表明,与同期A、B、C组比较,D组股骨头内骨坏死面积明显增多,骨小梁稀疏变细、断裂更明显,股骨头髓腔内脂肪细胞数量明显增多,空骨陷窝数目增多。
第二部分：48h后可见少数细胞贴壁,为成纤维细胞样细胞。4d后少量细胞呈长梭形,P3代贴壁细胞通过流式细胞仪检测,阳性表达CD44。贴壁细胞经BrdU标记后包埋于FS能存活,4天后细胞呈典型的梭形细胞形态,7天后生物蛋白胶边缘部分开始降解,细胞脱落致培养板；体外培养14天,细胞生长良好,大部分FS降解,脱落的细胞增多,贴壁生长的细胞形态正常；三周后生物蛋白胶完全降解,四组相比,细胞形态未受影响。P3代BMSCs经BrdU标记后在倒置显微镜下发现,细胞核内呈现棕红色,极少部分未着色,标记率达90%以上。孵育12h各组细胞活性均在90%以上,P>0.05,差异无统计学意义,孵育24、48、72h、各组细胞活性与12h相似。在MTT法检测生长动力学OD值、碱性磷酸酶及钙定量检测显示,B,C,D与A组(对照组)相比及B,C,D三组间相比,进行重复测量方差分析,P＞0.05,无统计学意义。
第三部分：B组(普伐他汀组)24h后光镜下BMSCs存活,4天后细胞呈典型的梭形细胞形态,7天后生物蛋白胶边缘部分开始降解,细胞脱落致培养板；体外培养14天,细胞生长良好,大部分生物蛋白胶降解,脱落的细胞增多,贴壁生长的细胞形态正常；ALP比活性及细胞外基质矿化能力明显强于A组(对照组),(P＜0.05)；Elisa法检测VEGF和BMP-2表达量高于A组,(P＜0.05)；基因组寡肽核苷酸芯片杂交,扫描分析2723个基因,共检测出326个差异基因表达,其中包括ALP1,BMP-2,OCN,DLX5, Cbfa1MMP13等与成骨分化有关的基因。
第四部分：血清胆固醇(CHOL)、甘油三脂(TG)、高密度脂蛋白(HDL)造模后4周4组组间比较无统计学差异(P＞0.05)。血清胆固醇(CHOL)、甘油三脂(TG)造模后8周D组与A、B、C三组比较含量较低,组间比较均有统计学差异(P<0.05)。A、B、C三组之间比较无统计学差异；造模后12周D组含量降低,其他三组也相对降低,B、C两组之间比较均无统计学差异(P＞0.05)。第16周D组含量明显比其他三组降低,与其他三组分别比较均有显著统计学差异(P＜0.01)。第8周高密度脂蛋白(HDL)的含量A、B、C三组随时间推移逐渐降低D组与之相反,第12、16周B、C两组组内比较均无统计学差异(P>0.05),D组与其他三组比较均有统计学差异(P<0.05)。病理学研究表明,D与同期A、B、C三组比较组股骨头内骨坏死面积明显减少,股骨头髓腔内脂肪细胞数量明显减少,空骨陷窝比率降低,骨小梁密度相对增高,骨组织内VEGF、Cbfa1的表达明显增强。实时荧光定量检测8周A、B、C、D四组VEGF、Cbfa1 mRNA的表达量无明显差异,但造模后12周及16周D组VEGF、Cbfa1 mRNA的表达量B、C、D三组都不同程度的增加其中D组改变的著明显,16周D组与B、C三组比较有统计学差异(P<0.05),与A组比较有显著统计学差异(P<0.01)。8周时MRI坏死区信号改变A组坏死区扩大,B组无扩大,C组范围缩小,D组明显缩小。12、16周A组MRI间明显的坏死信号改变,B、C两组坏死信号区域有所减小,D组坏死信号基木接近正常。
结论：
第一部分：
1.在相同的实验条件下,双次注射内毒素加三次注射激素造模出现时间最早、坏死效果最明显,骨陷窝空虚率随时间的延长而增加,为研究股骨头坏死提供较理想的实验平台。
2.造模后股骨头内微血管内皮细胞受损,骨内小动脉断裂迂曲、栓塞；血脂代谢紊乱,形成高脂血症；凝血功能异常,血液呈高凝状态,可能是导致早期激素性股骨头坏死的发生机制。
第二部分：
1.FS具有良好的组织相容性,可塑性和生物可降解性,并具有三维立体结构,是一种适用于微创操作的可注射型组织工程细胞培养和移植的支架材料。
2. BrdU标记简单,快速,安全,检测敏感性好,是反映细胞增殖及示踪监测移植细胞的理想指标。
3.经BrdU标记的BMSCs包埋于可注射型FS后构建的支架材料是一种具有良好生物相容性的工程化组织。
第三部分：
1.普伐他汀可促进体外诱导培养的BMSCs/FS向成骨细胞分化,其作用机制可能与调控多种成骨相关基因表达有关。
2.普伐他汀对体外诱导培养的BMSCs/FS分泌VEGF和BMP-2具有促进作用。
第四部分：
1、Brdu标记的BMSCs/FS经髓芯减压通道能有效的移植到股骨头,在骨内微环境中可以向成骨细胞、骨细胞分化,对股骨头坏死的再生修复起着促进作用。
2、普伐他汀可以改善体内脂质代谢紊乱,减轻激素性坏死股骨头内脂肪细胞对骨细胞的损害；上调坏死股骨头内VEGF的表达,改善局部血供；上调坏死股骨头内Cbfal的表达,诱发新骨形成。
3.普伐他汀全身应用与BMSCs局部移植相结合,对治疗兔早期激素性股骨头坏死具有可行性,有望成为临床上治疗早期激素性股骨头坏死的新方法。
Objective:To establish a stable model for steroid-induced avascular necrosis of femoral head(SANFH) in rabbits through twice injection of E.coli endotoxin(LPS) and three times injection of methylprednisolone(MPS), to explore the mechanism of the avascular necrosis of femoral head. To establish the engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded,we verify the tissular and cellular biocompatibility of the engineering tissue;to observe the effects of pravastatin in vitro on osteogenic differentiation ability and expression of VEGF、BMP-2 for BMSCs/FS complex; to observe the effect of the pravastatin for the SANFH with BMSCs-FS through decompression and we also explore the mechanism of the pravastatin for the SANFH.
Methods:
Chapter 1
To establish a stable model for steroid-induced avascular necrosis of femoral head(SANFH) in rabbits:80 New Zealand rabbits (both male and female available) were obtained,and then randomly divided into 4 groups.Group A(control group):rabbits were intramuscular injected with normal saline at the same time point as the same as group D;Group B:injection of horse sera and three times of methylprednisolone;Group C:injection of single E.coli endotoxin and three times of methylprednisolone;Group D:injection of twice E.coli endotoxin and three times of methylprednisolone. We observe the animal's general condition.and we also detect the level of cholesterol(CHOL)、triglycerides(TG)、blood rheology、coagulation state、thrombomodulin.All the data was analysed by SPSS 17.0.We used the T test to analyse the data between two sets and used the q test to analyse the data among multiple sets.We identified the statistical significant difference with P<0.01.We obtained the bilateral general femoral head sample after 6、8、10、12 weeks and we check the general pathological section through light microscopy and carry out immunohistochemical staining of vascular endothelial growth factor(VEGF) and microthrobus(MSB). we performed the X-ray、MRI to elucidate the medical imaging changes of the SANFH after 8、10、12 weeks.
Chapter 2
To establish the engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded:Isolating the BMSCs from the femoral marrow cavity of the New Zealand rabbits,purifying the BMSCs through the repeated medium changes and passaging by the adherence,and the we identified the BMSCs through CD34、CD44 test through FCM. We divided the P3 BMSCs into 4 groups.Group A:P3 BMSCs+osteoinduction medium,Group B:P3BMSCs+Brdu+osteoinduction medium,Group C: P3 BMSCs+fibrin glue+ osteoinduction medium,Group D:P3 BMSCs+fibrin glue+ Brdu+osteoinduction medium. On the 1、2、4、7、14、21 day after osteoinduction,we performed the cell kinetics tests、light microscopy observation trypan blue staining、calcium-cobalt staining and Von-Kossa staining and examined the amount of ALP and Ca quantitatively.We evaluated the data by SPSS 17.0.
Chapter 3
To observe the effects of pravastatin in vitro on osteogenic differentiation ability and expression of VEGF、BMP-2 for BMSCs/FS complex:P3 BMSCs+FS+osteogenic medium:group A(control),group B(100 umol/L pravatatin).Investigate the cell growth kinetics,observe the cell morphology under inverted microscope at day 1、4、7、14; check the ALP quantity at day 14;check the osteogenic ability using Von-Kossa staining at day 21;extract the mRNA,compound cDNA using reverse transcription,to hybrid the FITC-cDNA with genomic microarray chip,to discriminate the differentially expressed genes.To check the VEGF and BMP-2 expression.To analyse the data with SPSS 17.0.
Chapter 4
To observe the effect of the pravastatin for the SANFH with BMSCs-FS through decompression:60SANFH-rabbits were randomly divided into four groups(15each group).Group A:control;Group B:core decompression only;Group C:Brdu marked BMSCs only;Group D:pravastatin+Brdu marked BMSCs. We test the level of blood fat at 4、8、12、 16 weeks after operation.At 8、12、16 weeks after operation,rabbits were sacrificed,the femoral heads were observed through naked eye、light microscopy、electron microscope.We also examined expression of VEGF and Cbfa1 through real-time PCR at 8、12、16 weeks after operation.We examined the Brdu marked BMSCs in the femoral head at 8、12、16 week after operation and performed the MRI. To analyse the data with SPSS 17.0.
Results:
Chapter 1
At 4 week after the final injection,the rabbits'weight of group A increased and the rabbits' weight of group B、C、D decreased.The rabbits of group B、C、D showed listlessness、less activity、reduced diet and less response.4animals died at 10 days after final injection in group B,5 animals died at 10 days after final injection in group C,7 animals died at 10days after final injection in group D.Compared with group A,the level of CHOL、TG、the whole blood viscosity low shear rate、viscosity of plasma、erythrocyte aggregation index significantly increased(P<0.05),which group D increased more significantly.The MRI showed no change before injection and 2 week after injection.The signal intensity of MRI in group C and D decreased slightly and the signal intensity of MRI in group A and B at four week after injection.The MRI detected small necrotic area of femoral head in group C and D and the signal intensity of MRI in group B decreased slightly at 8 week after injection.The necrotic area of femoral head enlarged at 12 week after injection in group D.The rate of empty lacuna is 10.4%±4.4% in group B、19.7%±4.7 in group C、33.2%±5.6% in group D and the area of bone trabecula is 64.4%±8.4% in group B,53.2%±5.7% in group C, 41.3%±5.1% in group D.Compared with group B and C,the group D showed the statistical difference(P<0.01).Compared with group A、B、C on medical imaging and pathology, the necrotic area of femoral head、fat cells in intramedullary、empty lacuna in femoral head increased significantly in group D and the bone trabecula became thin and more fractured.
Chapter 2
Little cells can attach on the culturing bottle 48 hours later,presenting like fibroblast;most cells present long fusiform 4d later.The P3 BMSCs expressed CD44 through FCM. The Brdu marked BMSCs embedded in the fibrin glue can survive. The BMSCs presented like the fibroblast 3 days later;the brim of fibrin glue began to degrade 6 days later and the BMSCs fell onto the plat; the most of the fibrin glue degraded and more BMSCs fell onto the plate and the BMSCs presented the normal morphous 14 days later;all the fibrin glue degraded 21 days later and the fibrin displayed no bad effect on the morphous of BMSCs. More than 90% of Brdu marked P3 BMSCs showed the color of sepia in the nucleus under the light microscopy,more 90% of Brdu marked P3 BMSCs showed the color of sepia in the nucleus under the light microscopy at 12、24、48、72 hour after cultivation.there is no statistical differences in each group(P>0.05).The OD of MTT、quantity of ALP and Ca showed no statistical differences in each group(P>0.05).
Chapter 3
The Brdu marked BMSCs embedded in the the FS in group B can survive;the cells present typical fusiform 4d later;the margin of the fibrin glue began to degrade and the cells fell onto the plate 7d later; the BMSCs proliferate good and most FS degradated;more and more BMSCs fell onto the plate and the morphology of adherent cells presented normal 14d later. The quantity of ALP in group B are more than that in group A(P<0.05) and the pravastatin can upregulate the VEGF and BMP2 through ELISA(P<0.05). Among 2723 genes,there are 326 differentially expressed genes including ALP1,BMP-2,OCN,DLX5, Cbfa1MMP13 which are closely correlated with osteogenic differentiation through the microarray chip.
Chapter 4
The level of CHOL、TG、HDL showed no statistical differences in each group(P>0.05) at 1 week after operation. The level of CHOL、TG in group D decreased and had statistical difference compared with other groups.there is no no statistical difference among group A、B、C at 4 week after operation. The level of CHOL、TG in group D decreased significantly and other groups decreased slightly at 8 week after operation and is no no statistical difference between group B and C(P>0.05). At 12 week after operation,the level of CHOL、TG in group D decreased more significantly and had statistical difference compared with other groups(P<0.01). The level of HDL in group A、B、C decreased,but increased in group D at 4 week after operation.At 8、12 week after operation,there were no statistical differences between group B and C(P>0.05),but there were statistical differences in group D compared with other groups.By pathological examination,the necrotic area of femoral head、fat cells in intramedullary、the rate of empty lacuna decreased but the density of bone trabecular increased in group D compared with other groups.The expression of VEGF、Cbfa1 showed no differences in each group through real-time PCR at 4 week after operation. At 8 week after operation,the expression of VEGF、Cbfa1 upregulated in all groups but group D upregulated more significantly. At 16 week after operation,the group showed the statistical differences compared with other groups(P<0.05).The necrotic area of femoral head enlarged in group A,no changing in group B,decreased in group C,decreasing significantly in group D at 4 week after operation. The necrotic area of femoral head enlarged slightly in group A,decreasing slightly in group B and C,disappearing almostly in group D.
Conclusion:
Chapter 1
1. Under the identical experimental condition,the appearance of SANFH were the earliest and most obvious in the group which receive the twice injection of E.coli endotoxin and thrice injection of glucocorticoid and the empty lacuna increased day by day.lt can provide good animal model of SANFH for the clinical research.
2. The micrangium endothelial cells in the necrotic femoral head were impaired after the rabbits SANFH model made.The arteriolae in the femoral head were impaired and thrombosis;also resulted in the dyslipidemia; and also resulted in dysfunction of blood coagulation and hypercoagulative state.This may underlie the pathological mechanism of early stage SANFH.
Chapter 2
1. The biological fibrin glue are satisfactory compatible、plastical and biological degradable,it has trhee-dimensional structure.lt can be used as injectable scaffold for cell cultivation and transplant.
2. The use of Brdu for marking is easy、swift、safe、sensitive,and it can reflect the proliferation and position of BMSCs.
3. The engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded present excellent biocompatibility
Chapter 3
1.The pravastatin can induce the BMSCs to differentiate to osteoblast and the mechanism maybe correlate to upregulate the osteogenic genes expressions.
2.The pravatatin can induce the BMSCs/FS to secrete the VEGF and BMP2.
Chapter 4
1. The BMSCs combined with FS can be transplanted into the femoral head through decompression,and the BMSCs can differentiated into osteoblast and osteocyte and it can help repair and regenerate the bone tissue of the SANFH.
2. Pravastatin can improve the lipid metabolism in vivo and ameliorate the toxic effect of the adipocyte on the osteocyte.It can upregulate the VEGF expression in the femoral head and improve the blood supply in the femoral head.It can also upregulate the Cbfal expression in the femoral head and help form new bone.
3. The combination of the systemic application of pravastatin and local application of BMSCs for curing SANFH are feasible and can be hoped to apply clinically in the therapy for early-stage SANFH.
双次注射内毒素加三次注射激素建立兔早期激素性股骨头坏死模型,探讨其可能的发病机制；构建可注射型FS包埋Brdu标记的BMSCs的工程化组织,验证其生物相容性以及体外成骨诱导分化的效果；观察普伐他汀在体外对BMSCs/FS成骨诱导分化及分泌VEGF, BMP-2的影响；观察普伐他汀对BMSCs/FS经髓芯减压移植治疗兔早期激素性股骨头坏死的干预效果,并探讨其作用机理。
方法：
第一部分：兔早期激素性股骨头坏死模型建立：取健康成年新西兰大白兔80只,雌雄不限,随机分成4组,每组各20只。A组：(对照组)与D组相同时间点注射等量生理盐水。B组：马血清加三次注射激素组；C组：单次注射内毒素加三次注射激素组；D组：双次注射内毒素加三次注射激素组。注射后观察动物一般情况。各组于用药后第4、6、8、10、12周行血清胆固醇、甘油三酯,血液流变学、凝血功能,血栓调节蛋白含量等检测,并应用SPSS17.0版统计软件,两组数据间比较采用t检验,多组数据间比较则采用单因素方差分析,进行q检验,以P<0.01作为统计学具有显著性差异的标准对数据进行分析处理。于第6、8、10、12周处死兔子取双侧股骨头行大体标本、常规组织病理学切片及血管内皮生长因子免疫组化染色、股骨头微血管内血栓染色,于注射前及末次注射后4、8及12周,行X线片、MRI检查,观察股骨头影像学改变。
第二部分：构建可注射型FS包埋Brdu标记的BMSCs的工程化组织：取健康新西兰大白兔8只,雌雄不限,采用全骨髓培养法,经过换液及传代,贴壁生长细胞通过流式细胞仪检测CD34、CD44等表面标记物,证实为高纯度的BMSCs后；取P3代BMSCs,分为四组：A组(对照组)：BMSCs+成骨培养基培养；B组(BrdU组)BMSCs +BrdU+成骨培养基培养；C组(生物蛋白胶组)：BMSCs+生物蛋白胶+成骨培养基培养；D组(生物蛋白胶+BrdU组)：BMSCs+生物蛋白胶+BrdU+成骨培养基避光培养。成骨诱导1、2、4、7、14、21天后,分别行光镜下大体观察,生长动力学检测、台盼蓝染色、碱性磷酸酶Gomori钙钴法和Von-Kossa改良法染色鉴定向成骨方向分化的能力,检测碱性磷酸酶定量和游离钙定量检测(甲基百里香酚蓝比色法),采用SPSS17.0统计软件对数据进行统计分析。
第三部分：观察普伐他汀在体外对BMSCs/FS成骨诱导分化及分泌VEGF,BMP-2的影响：BrdU标记的P3代BMSCs/FS加入成骨培养基避光培养,设对照组(A组)：加入等量溶剂,实验组(B组)：加入普伐他汀(100umol/L)。细胞培养1、4、7、14天在倒置显微镜下观察细胞形态,进行生长动力学检测,14天碱性磷酸酶(alkaline phosphatase,ALP)活性测定,21天Von-Kossa改良法染色鉴定向成骨方向分化的能力,第21天提取纯化mRNA,逆转录合成cDNA,荧光标记后与全基因组寡核苷酸芯片杂交,扫描后筛选出差异表达基因。第14天采用Elisa法检测VEGF和BMP-2表达量。采用SPSS17.0统计软件对数据进行统计分析。
第四部分：观察普伐他汀对BMSCs/FS经髓芯减压移植治疗兔早期激素性股骨头坏死的干预效果：取造模4周后存活的成年新西兰白兔60只,雌雄不限,随机分为四组每组15只：A组：(对照组)不做任何处理；B组：(髓芯减压组)C臂X光透视下用骨穿针行股骨头髓芯减压；C组：(干细胞移植组)股骨头髓芯减压同时局部移植Brdu标记的BMSCs/FS, D组：(干细胞移植联合普伐他汀治疗组)在C组基础上次日起以普伐他汀灌胃。造模后4周、8周、12周及16周,抽取耳缘静脉血检测血脂。造模后8周、12周、16周分批处死兔子,截取双侧股骨头,分别进行大体观察、常规组织病理学切片光镜和电镜观察。8周、12周及16周行血管内皮生长因子、核心结合因子免疫组化染色,8周、12周及16周实时荧光定量检测VEGF和Cbfa1mRNA的表达。造模后8周C、D两组行Brdu标记干细胞示踪检测。造模后8周、12周及16周,行双侧股骨头1.5T核磁共振(MRI)检测观察坏死信号的改变。采用SPSS17.0统计软件对数据进行统计分析。
结果：
第一部分：A组正常,B、C、D组动物于末次注射后3周体重减轻,精神萎靡,活动及饮食减少,反应淡漠,分别有4、5、7只动物死亡,各期血清胆固醇、甘油三酯、低切变率下全血黏度、血浆黏度、红细胞聚集指数均明显升高(P＜0.05),以D组更为明显。自造模后10周始,两组间各指标比较均有统计学差异((P＜0.05)。注射前及注射后2周各组MRI显示正常,C、D组4周股骨头信号轻度减低,A、B组未见异常。C、D组8周股骨头有小部分坏死区域,B组信号轻度减低,12周后D组股骨头坏死区域进一步扩大。B、C、D组空骨陷窝率分别为10.4%±4.4%,19.7%±4.7,33.2%±5.6%。骨小梁面积百分比分别为64.4%±8.4%,53.2%±±5.7%,41.3%±5.1%；D组指标与B,C组比较,差异有显著统计学意义(P<0.01)。影像学及病理学研究表明,与同期A、B、C组比较,D组股骨头内骨坏死面积明显增多,骨小梁稀疏变细、断裂更明显,股骨头髓腔内脂肪细胞数量明显增多,空骨陷窝数目增多。
第二部分：48h后可见少数细胞贴壁,为成纤维细胞样细胞。4d后少量细胞呈长梭形,P3代贴壁细胞通过流式细胞仪检测,阳性表达CD44。贴壁细胞经BrdU标记后包埋于FS能存活,4天后细胞呈典型的梭形细胞形态,7天后生物蛋白胶边缘部分开始降解,细胞脱落致培养板；体外培养14天,细胞生长良好,大部分FS降解,脱落的细胞增多,贴壁生长的细胞形态正常；三周后生物蛋白胶完全降解,四组相比,细胞形态未受影响。P3代BMSCs经BrdU标记后在倒置显微镜下发现,细胞核内呈现棕红色,极少部分未着色,标记率达90%以上。孵育12h各组细胞活性均在90%以上,P>0.05,差异无统计学意义,孵育24、48、72h、各组细胞活性与12h相似。在MTT法检测生长动力学OD值、碱性磷酸酶及钙定量检测显示,B,C,D与A组(对照组)相比及B,C,D三组间相比,进行重复测量方差分析,P＞0.05,无统计学意义。
第三部分：B组(普伐他汀组)24h后光镜下BMSCs存活,4天后细胞呈典型的梭形细胞形态,7天后生物蛋白胶边缘部分开始降解,细胞脱落致培养板；体外培养14天,细胞生长良好,大部分生物蛋白胶降解,脱落的细胞增多,贴壁生长的细胞形态正常；ALP比活性及细胞外基质矿化能力明显强于A组(对照组),(P＜0.05)；Elisa法检测VEGF和BMP-2表达量高于A组,(P＜0.05)；基因组寡肽核苷酸芯片杂交,扫描分析2723个基因,共检测出326个差异基因表达,其中包括ALP1,BMP-2,OCN,DLX5, Cbfa1MMP13等与成骨分化有关的基因。
第四部分：血清胆固醇(CHOL)、甘油三脂(TG)、高密度脂蛋白(HDL)造模后4周4组组间比较无统计学差异(P＞0.05)。血清胆固醇(CHOL)、甘油三脂(TG)造模后8周D组与A、B、C三组比较含量较低,组间比较均有统计学差异(P<0.05)。A、B、C三组之间比较无统计学差异；造模后12周D组含量降低,其他三组也相对降低,B、C两组之间比较均无统计学差异(P＞0.05)。第16周D组含量明显比其他三组降低,与其他三组分别比较均有显著统计学差异(P＜0.01)。第8周高密度脂蛋白(HDL)的含量A、B、C三组随时间推移逐渐降低D组与之相反,第12、16周B、C两组组内比较均无统计学差异(P>0.05),D组与其他三组比较均有统计学差异(P<0.05)。病理学研究表明,D与同期A、B、C三组比较组股骨头内骨坏死面积明显减少,股骨头髓腔内脂肪细胞数量明显减少,空骨陷窝比率降低,骨小梁密度相对增高,骨组织内VEGF、Cbfa1的表达明显增强。实时荧光定量检测8周A、B、C、D四组VEGF、Cbfa1 mRNA的表达量无明显差异,但造模后12周及16周D组VEGF、Cbfa1 mRNA的表达量B、C、D三组都不同程度的增加其中D组改变的著明显,16周D组与B、C三组比较有统计学差异(P<0.05),与A组比较有显著统计学差异(P<0.01)。8周时MRI坏死区信号改变A组坏死区扩大,B组无扩大,C组范围缩小,D组明显缩小。12、16周A组MRI间明显的坏死信号改变,B、C两组坏死信号区域有所减小,D组坏死信号基木接近正常。
结论：
第一部分：
1.在相同的实验条件下,双次注射内毒素加三次注射激素造模出现时间最早、坏死效果最明显,骨陷窝空虚率随时间的延长而增加,为研究股骨头坏死提供较理想的实验平台。
2.造模后股骨头内微血管内皮细胞受损,骨内小动脉断裂迂曲、栓塞；血脂代谢紊乱,形成高脂血症；凝血功能异常,血液呈高凝状态,可能是导致早期激素性股骨头坏死的发生机制。
第二部分：
1.FS具有良好的组织相容性,可塑性和生物可降解性,并具有三维立体结构,是一种适用于微创操作的可注射型组织工程细胞培养和移植的支架材料。
2. BrdU标记简单,快速,安全,检测敏感性好,是反映细胞增殖及示踪监测移植细胞的理想指标。
3.经BrdU标记的BMSCs包埋于可注射型FS后构建的支架材料是一种具有良好生物相容性的工程化组织。
第三部分：
1.普伐他汀可促进体外诱导培养的BMSCs/FS向成骨细胞分化,其作用机制可能与调控多种成骨相关基因表达有关。
2.普伐他汀对体外诱导培养的BMSCs/FS分泌VEGF和BMP-2具有促进作用。
第四部分：
1、Brdu标记的BMSCs/FS经髓芯减压通道能有效的移植到股骨头,在骨内微环境中可以向成骨细胞、骨细胞分化,对股骨头坏死的再生修复起着促进作用。
2、普伐他汀可以改善体内脂质代谢紊乱,减轻激素性坏死股骨头内脂肪细胞对骨细胞的损害；上调坏死股骨头内VEGF的表达,改善局部血供；上调坏死股骨头内Cbfal的表达,诱发新骨形成。
3.普伐他汀全身应用与BMSCs局部移植相结合,对治疗兔早期激素性股骨头坏死具有可行性,有望成为临床上治疗早期激素性股骨头坏死的新方法。
Objective:To establish a stable model for steroid-induced avascular necrosis of femoral head(SANFH) in rabbits through twice injection of E.coli endotoxin(LPS) and three times injection of methylprednisolone(MPS), to explore the mechanism of the avascular necrosis of femoral head. To establish the engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded,we verify the tissular and cellular biocompatibility of the engineering tissue;to observe the effects of pravastatin in vitro on osteogenic differentiation ability and expression of VEGF、BMP-2 for BMSCs/FS complex; to observe the effect of the pravastatin for the SANFH with BMSCs-FS through decompression and we also explore the mechanism of the pravastatin for the SANFH.
Methods:
Chapter 1
To establish a stable model for steroid-induced avascular necrosis of femoral head(SANFH) in rabbits:80 New Zealand rabbits (both male and female available) were obtained,and then randomly divided into 4 groups.Group A(control group):rabbits were intramuscular injected with normal saline at the same time point as the same as group D;Group B:injection of horse sera and three times of methylprednisolone;Group C:injection of single E.coli endotoxin and three times of methylprednisolone;Group D:injection of twice E.coli endotoxin and three times of methylprednisolone. We observe the animal's general condition.and we also detect the level of cholesterol(CHOL)、triglycerides(TG)、blood rheology、coagulation state、thrombomodulin.All the data was analysed by SPSS 17.0.We used the T test to analyse the data between two sets and used the q test to analyse the data among multiple sets.We identified the statistical significant difference with P<0.01.We obtained the bilateral general femoral head sample after 6、8、10、12 weeks and we check the general pathological section through light microscopy and carry out immunohistochemical staining of vascular endothelial growth factor(VEGF) and microthrobus(MSB). we performed the X-ray、MRI to elucidate the medical imaging changes of the SANFH after 8、10、12 weeks.
Chapter 2
To establish the engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded:Isolating the BMSCs from the femoral marrow cavity of the New Zealand rabbits,purifying the BMSCs through the repeated medium changes and passaging by the adherence,and the we identified the BMSCs through CD34、CD44 test through FCM. We divided the P3 BMSCs into 4 groups.Group A:P3 BMSCs+osteoinduction medium,Group B:P3BMSCs+Brdu+osteoinduction medium,Group C: P3 BMSCs+fibrin glue+ osteoinduction medium,Group D:P3 BMSCs+fibrin glue+ Brdu+osteoinduction medium. On the 1、2、4、7、14、21 day after osteoinduction,we performed the cell kinetics tests、light microscopy observation trypan blue staining、calcium-cobalt staining and Von-Kossa staining and examined the amount of ALP and Ca quantitatively.We evaluated the data by SPSS 17.0.
Chapter 3
To observe the effects of pravastatin in vitro on osteogenic differentiation ability and expression of VEGF、BMP-2 for BMSCs/FS complex:P3 BMSCs+FS+osteogenic medium:group A(control),group B(100 umol/L pravatatin).Investigate the cell growth kinetics,observe the cell morphology under inverted microscope at day 1、4、7、14; check the ALP quantity at day 14;check the osteogenic ability using Von-Kossa staining at day 21;extract the mRNA,compound cDNA using reverse transcription,to hybrid the FITC-cDNA with genomic microarray chip,to discriminate the differentially expressed genes.To check the VEGF and BMP-2 expression.To analyse the data with SPSS 17.0.
Chapter 4
To observe the effect of the pravastatin for the SANFH with BMSCs-FS through decompression:60SANFH-rabbits were randomly divided into four groups(15each group).Group A:control;Group B:core decompression only;Group C:Brdu marked BMSCs only;Group D:pravastatin+Brdu marked BMSCs. We test the level of blood fat at 4、8、12、 16 weeks after operation.At 8、12、16 weeks after operation,rabbits were sacrificed,the femoral heads were observed through naked eye、light microscopy、electron microscope.We also examined expression of VEGF and Cbfa1 through real-time PCR at 8、12、16 weeks after operation.We examined the Brdu marked BMSCs in the femoral head at 8、12、16 week after operation and performed the MRI. To analyse the data with SPSS 17.0.
Results:
Chapter 1
At 4 week after the final injection,the rabbits'weight of group A increased and the rabbits' weight of group B、C、D decreased.The rabbits of group B、C、D showed listlessness、less activity、reduced diet and less response.4animals died at 10 days after final injection in group B,5 animals died at 10 days after final injection in group C,7 animals died at 10days after final injection in group D.Compared with group A,the level of CHOL、TG、the whole blood viscosity low shear rate、viscosity of plasma、erythrocyte aggregation index significantly increased(P<0.05),which group D increased more significantly.The MRI showed no change before injection and 2 week after injection.The signal intensity of MRI in group C and D decreased slightly and the signal intensity of MRI in group A and B at four week after injection.The MRI detected small necrotic area of femoral head in group C and D and the signal intensity of MRI in group B decreased slightly at 8 week after injection.The necrotic area of femoral head enlarged at 12 week after injection in group D.The rate of empty lacuna is 10.4%±4.4% in group B、19.7%±4.7 in group C、33.2%±5.6% in group D and the area of bone trabecula is 64.4%±8.4% in group B,53.2%±5.7% in group C, 41.3%±5.1% in group D.Compared with group B and C,the group D showed the statistical difference(P<0.01).Compared with group A、B、C on medical imaging and pathology, the necrotic area of femoral head、fat cells in intramedullary、empty lacuna in femoral head increased significantly in group D and the bone trabecula became thin and more fractured.
Chapter 2
Little cells can attach on the culturing bottle 48 hours later,presenting like fibroblast;most cells present long fusiform 4d later.The P3 BMSCs expressed CD44 through FCM. The Brdu marked BMSCs embedded in the fibrin glue can survive. The BMSCs presented like the fibroblast 3 days later;the brim of fibrin glue began to degrade 6 days later and the BMSCs fell onto the plat; the most of the fibrin glue degraded and more BMSCs fell onto the plate and the BMSCs presented the normal morphous 14 days later;all the fibrin glue degraded 21 days later and the fibrin displayed no bad effect on the morphous of BMSCs. More than 90% of Brdu marked P3 BMSCs showed the color of sepia in the nucleus under the light microscopy,more 90% of Brdu marked P3 BMSCs showed the color of sepia in the nucleus under the light microscopy at 12、24、48、72 hour after cultivation.there is no statistical differences in each group(P>0.05).The OD of MTT、quantity of ALP and Ca showed no statistical differences in each group(P>0.05).
Chapter 3
The Brdu marked BMSCs embedded in the the FS in group B can survive;the cells present typical fusiform 4d later;the margin of the fibrin glue began to degrade and the cells fell onto the plate 7d later; the BMSCs proliferate good and most FS degradated;more and more BMSCs fell onto the plate and the morphology of adherent cells presented normal 14d later. The quantity of ALP in group B are more than that in group A(P<0.05) and the pravastatin can upregulate the VEGF and BMP2 through ELISA(P<0.05). Among 2723 genes,there are 326 differentially expressed genes including ALP1,BMP-2,OCN,DLX5, Cbfa1MMP13 which are closely correlated with osteogenic differentiation through the microarray chip.
Chapter 4
The level of CHOL、TG、HDL showed no statistical differences in each group(P>0.05) at 1 week after operation. The level of CHOL、TG in group D decreased and had statistical difference compared with other groups.there is no no statistical difference among group A、B、C at 4 week after operation. The level of CHOL、TG in group D decreased significantly and other groups decreased slightly at 8 week after operation and is no no statistical difference between group B and C(P>0.05). At 12 week after operation,the level of CHOL、TG in group D decreased more significantly and had statistical difference compared with other groups(P<0.01). The level of HDL in group A、B、C decreased,but increased in group D at 4 week after operation.At 8、12 week after operation,there were no statistical differences between group B and C(P>0.05),but there were statistical differences in group D compared with other groups.By pathological examination,the necrotic area of femoral head、fat cells in intramedullary、the rate of empty lacuna decreased but the density of bone trabecular increased in group D compared with other groups.The expression of VEGF、Cbfa1 showed no differences in each group through real-time PCR at 4 week after operation. At 8 week after operation,the expression of VEGF、Cbfa1 upregulated in all groups but group D upregulated more significantly. At 16 week after operation,the group showed the statistical differences compared with other groups(P<0.05).The necrotic area of femoral head enlarged in group A,no changing in group B,decreased in group C,decreasing significantly in group D at 4 week after operation. The necrotic area of femoral head enlarged slightly in group A,decreasing slightly in group B and C,disappearing almostly in group D.
Conclusion:
Chapter 1
1. Under the identical experimental condition,the appearance of SANFH were the earliest and most obvious in the group which receive the twice injection of E.coli endotoxin and thrice injection of glucocorticoid and the empty lacuna increased day by day.lt can provide good animal model of SANFH for the clinical research.
2. The micrangium endothelial cells in the necrotic femoral head were impaired after the rabbits SANFH model made.The arteriolae in the femoral head were impaired and thrombosis;also resulted in the dyslipidemia; and also resulted in dysfunction of blood coagulation and hypercoagulative state.This may underlie the pathological mechanism of early stage SANFH.
Chapter 2
1. The biological fibrin glue are satisfactory compatible、plastical and biological degradable,it has trhee-dimensional structure.lt can be used as injectable scaffold for cell cultivation and transplant.
2. The use of Brdu for marking is easy、swift、safe、sensitive,and it can reflect the proliferation and position of BMSCs.
3. The engineering tissue made by fibrin sealant where the Brdu marked bone marrow mesenchymal stem cells(BMSCs) embedded present excellent biocompatibility
Chapter 3
1.The pravastatin can induce the BMSCs to differentiate to osteoblast and the mechanism maybe correlate to upregulate the osteogenic genes expressions.
2.The pravatatin can induce the BMSCs/FS to secrete the VEGF and BMP2.
Chapter 4
1. The BMSCs combined with FS can be transplanted into the femoral head through decompression,and the BMSCs can differentiated into osteoblast and osteocyte and it can help repair and regenerate the bone tissue of the SANFH.
2. Pravastatin can improve the lipid metabolism in vivo and ameliorate the toxic effect of the adipocyte on the osteocyte.It can upregulate the VEGF expression in the femoral head and improve the blood supply in the femoral head.It can also upregulate the Cbfal expression in the femoral head and help form new bone.
3. The combination of the systemic application of pravastatin and local application of BMSCs for curing SANFH are feasible and can be hoped to apply clinically in the therapy for early-stage SANFH.
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