可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损

背景：自体髂骨移植一直被认为是骨缺损修复的“金标准”,但其来源有限。 目的：验证应用可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损的效果。 方法：80只雄性SD大鼠建立双侧颅骨缺损模型,随机分为3组：骨修复材料+骨碎补总黄酮组采用可注射骨修复材料结合骨碎补总黄酮灌胃修复大鼠颅骨缺损;骨修复材料+去离子水组采用可注射骨修复材料结合去离子水灌胃修复大鼠颅骨缺损;羟基磷灰石+去离子水组采用羟基磷灰石结合去离子水灌胃修复大鼠颅骨缺损,1次/d,持续8周。于建模后2,4,8周取颅骨标本进行苏木精-伊红染色和Masson染色组织学观察。 结果与结论：羟基磷灰石组新骨形成和材料降解速度较慢;可注射骨修复材料组新骨形成和材料降解较羟基磷灰石组快,利于血管及纤维组织长入;骨碎补总黄酮灌胃可以促进血管及纤维组织长入材料,促进成骨。与羟基磷灰石相比,可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损,可促进新骨形成,缩短骨缺损修复时间。     

不同浓度骨碎补总黄酮复合可注射骨修复材料对MC3T3-E1成骨细胞作用的实验研究

目的本实验旨在观察中药骨碎补提取物骨碎补总黄酮(AFDR)复合海藻酸基可注射骨修复材料对成骨细胞MC3T3-E1体外培养的影响。方法将成骨细胞株MC3T3-E1随机分为空白对照组、单纯使用海藻酸基可注射骨修复材料组、AFDR0.04mg/L、0.16mg/L、0.64mg/L、1.28mg/L、5.12mg/L 7组,体外复合海藻酸基可注射骨修复材料,以24h和48h为观察时间点,倒置显微镜观察MC3T3-E1细胞形态,台盼蓝拒染法检测MC3T3-E1存活率,MTT法观察细胞增殖效应,碱性磷酸酶(ALP)活性和骨钙素定量检测分别观察不同浓度骨碎补总黄酮促进MC3T3-E1分化作用,采用Von kossa钙化染色法观察其促MC3T3-E1细胞钙化作用。结果①倒置显微镜下观察复合海藻酸基可注射骨修复材料的MC3T3-E1细胞形态,细胞形态呈三角形、多边形、长梭形等,呈集落样生长,无接触抑制,有伪足伸出,胞液透亮,核圆形。AFDR各组不同程度促进成骨细胞的增殖、分化:7组能不同程度的促进成骨细胞的增殖、分化,与空白对照组比较,以0.16 mg/L和0.64mg/L剂量组的成骨细胞增殖数量最多,1.28mg/L,5.12mg/L的AFDR对细胞增殖作用不明显;②MC3T3-E1平均存活率≥90%;③骨碎补总黄酮(AFDR)0.16 mg/L和0.64mg/L剂量组在培养24小时和48小时,其OD值与空白对照组及组间均有显著性差异,可促进MC3T3-E1细胞增殖(P0.05和P0.01);④AFDR 0.16 mg/L和0.64mg/L剂量组能使MC3T3-E1的ALP活性升高(P0.05);⑤AFDR 0.64mg/L剂量组能促进MC3T3-E1骨钙素合成(P0.05);⑥AFDR 0.16 mg/L和0.64mg/L剂量组均可促进细胞钙化(P0.05)。结论 AFDR可显著促进在海藻酸基可注射骨修复材料上MC3T3-E1的增殖、分化和钙化,其作用具有时间依赖和剂量依赖关系。     

新型仿生骨材料修复山羊颅骨缺损的实验研究

目的设计山羊颅骨缺损模型以验证自主研发的两种不同配比胶原基仿生骨材料的生物安全性和诱导成骨效果。方法首先使用环钻在山羊颅骨上制作圆形缺损区,再将胶原配比不同的仿生骨材料植入缺损区,观察材料植入后的炎症反应及缺损区域成骨情况等。结果胶原蛋白含量高的A组材料大部分降解且部分诱导成骨,胶原蛋白含量低的B组材料基本不降解且无诱导成骨效果。两组材料植入山羊体内后,均未出现炎症反应,说明自主研发的胶原基仿生骨材料的生物相容性良好。结论自主研发的胶原基仿生骨材料生物相容性和降解性良好,有一定的诱导成骨效果。     

骨碎补总黄酮干预Notch信号通路影响骨重建过程中成血管-成骨耦联

背景:骨缺损是骨科临床的难题,骨碎补总黄酮对骨缺损治疗有确切疗效,但是其具体机制尚不明确。目的:基于Notch信号通路探讨骨碎补总黄酮在骨重建过程中对血管生成和骨形成的影响。方法:48只SD大鼠随机分为模型组、中药组、中药+DAPT组、DAPT组,每组12只。构建大鼠股骨缺损模型,分别给予中药骨碎补总黄酮和Notch通路阻滞剂DAPT干预,干预12周后通过X射线片、Micro-CT和苏木精-伊红染色观察骨缺损的成骨情况,免疫组化检测骨组织中血管内皮生长因子、CD31、Hes1、骨形态发生蛋白2、Notch1蛋白的表达。结果与结论:①X射线片、Micro-CT和苏木精-伊红染色显示中药组的成骨效果明显优于其他3组;②免疫组化检测显示中药组骨形态发生蛋白2、血管内皮生长因子、CD31、Hes1、Notch1平均吸光度值较模型组明显增多(P<0.05);中药+DAPT组血管内皮生长因子、Hes1、骨形态发生蛋白2的平均吸光度值大于DAPT组(P<0.05),但CD31和Notch1表达组间差异无显著性意义;③结果表明,骨碎补总黄酮通过激活Notch通路促进骨重建过程中成血管-成骨耦联,从而促进骨修复。     

体外构建复合软质再生颅骨修复材料填充修复颅骨缺损

BACKGROUND: In the process of bone defect repair, as their respective shortcomings, both autograft and allograft cannot obtain satisfactory outcomes.div> OBJECTIVE: To observe the effect of soft composite materials for bone regeneration established in vitro in skull defect repair.     

注射型纳米羟基磷灰石/聚酰胺生物活性骨修复材料的研究

利用羟基磷灰石纳米晶体与聚酰胺66复合，构成新型生物活性骨修复材料，探讨用于不规则骨缺损修复的注射型纳米复合人工骨的生物学特性及骨组织修复能力。对该材料进行X射线衍射分析、凝结时间、凝结强度等研究及动物实验研究，评价该材料的组织相容性和缺损骨组织的修复能力。结果表明该材料的X射线衍射谱与羟基磷灰石／聚酰胺复合材料的X射线衍射谱相同；液固比为0．5时复合材料易于注射；在生理盐水或血液中的凝固时间为25～30min；在生理盐水中固化48h后，抗压强度为37MPa。植入后牙槽嵴表面软组织愈合良好，实验侧牙槽嵴修复高度明显大于对照侧；组织形态学观察，4周时材料周围未见有成骨迹象，16周时材料被包裹并在与其相连的区域出现成骨早期的片状结缔组织。研究证实，以一定的复合比例构成的纳米羟基磷灰石／聚酰胺66复合材料组织相容性良好，可以注射方式实现对不规则骨缺损的修复。     

PDLLA/PLA-PEG-PLA新型人工骨修复恒河猴颅骨缺损实验研究

目的　评估PDLLA/PLA-PEG-PLA在骨组织工程中的应用前景。 方法　在4只恒河猴双侧顶骨24个10 mm颅骨去骨膜全层骨缺损中分别植入4种筛选出的支架/成骨细胞复合体,术后6、　12周采用X线、组织学及扫描电镜观察评价骨缺损修复情况。 结果　大体观察,都未引起明显的全身反应,局部切口愈合良好。X线、组织学及扫描电镜均证实人工骨在术后6周、12周成骨能力明显高于空白对照组,也高于单纯注射成骨细胞对照组,而改性的PDLLA/PLA-PEG-PLA、PDLLA/β-TCP及　PDLLA/BG成骨能力优于单纯PDLLA,X线评分证实PDLLA/PLA-PEG-PLA成骨能力最强,12周时成骨量达缺损区70%。 结论　4种筛选的支架与成骨细胞复合构建组织工程人工骨具有较强的体内成骨效能,PDLLA/PLA-PEG-PLA在骨组织工程中有很好的应用前景。     

重组人骨形态发生蛋白2/骨修复材料的制备与性能

BACKGROUND: It has become a hotspot to prepare the bone repair material that exhibits natural bone structure and is used in combination with biological factors. OBJECTIVE: To prepare the recombinant human bone morphogenetic protein-2 (rhBMP-2)/bone repair material, and to evaluate its capacities of release, activity and ectopic osteoinduction. METHODS: A collagen-binding domain was added to the N-terminal of native rhBMP-2 that allowed bind to collagens in the bone repair material. Then, rhBMP-2/bone repair material was obtained through freeze-dried method. The releasing ability of rhBMP-2 in vitro was assayed by ELISA. C2C12 cell lines were loaded to the composite material with 0.25, 0.5 and 1 µg rhBMP-2, respectively. Afterwards, alkaline phosphatase activity was detected at 72 hours. The composite materials with 0, 2, 5 and 10 µg rhBMP-2 were implanted into the quadriceps of Sprague-Dawley rats, respectively. Alkaline phosphatase activity and the newly formed bone were detected at 2 and 4 weeks after implantation. The CY-7-labeled composite material was implanted into the quadriceps of Sprague-Dawley rats to observe its stability. RESULTS AND CONCLUSION: Substantially no rhBMP-2 from the rhBMP-2/bone repair material was released within 45 days. The alkaline phosphatase activity of C2C12 was in a rise with the increased concentration of rhBMP-2. The stability of the composite material in vivo was better, the alkaline phosphatase activity and ectopic bone formation increased as the concentration of rhBMP-2 rose. To conclude, the rhBMP-2/bone repair material preserves the stability of rhBMP-2, and improves ectopic osteoinduction ability.     

磷酸钙骨水泥与骨形态蛋白复合材料修复下颌骨缺损的研究

为提高磷酸钙骨水泥的成骨活性，将自制的磷酸钙骨水泥作为骨形态蛋白．2的载体予以复合，对复合载体材料进行物性研究和异位成骨试验。并以该复合材料修复兔下颌骨缺损，通过生物力学检测和骨界面新生骨计量，观察其修复下颌骨缺损的效果。结果表明，以该复合材料修复兔下颌骨缺损，其材料与宿主骨界面的结合强度以及新生骨量均明显高于单纯材料的修复。     

缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景：重组人骨形态发生蛋白2在体内半衰期短、易降解代谢,达不到理想的骨再生效果。 目的：制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料,并观察其缓释性能、骨诱导活性。 方法：将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜,涂覆于生物骨修复材料表面,ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成,观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内,2周后检测新生骨Ca2+离子含量,评价其异位骨诱导能力。 结果与结论：材料表面的壳聚糖膜分布均匀,负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释,前4 d释放量达总药量的50%,持续至12 d,释药量达到90%,第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比,缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强(P < 0.05)。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好,促进骨形成能力强。     

柚皮苷-壳聚糖/羟基磷灰石复合支架修复大鼠颅骨缺损

背景:骨碎补的有效成分柚皮苷具有补肝肾强筋骨的传统功效,能增加骨痂厚度,提高骨折愈合质量。目的:探究载中药骨碎补有效成分柚皮苷壳聚糖/羟基磷灰石复合支架的骨传导和骨诱导性能。方法:将一定钙磷比的羟基磷灰石前体液与含柚皮苷的壳聚糖溶液在碱性条件下原位结晶、冷冻干燥,获得柚皮苷-壳聚糖/羟基磷灰石多孔支架。将15只成年SD大鼠随机分成空白组(n=5)、对照组(n=5)和实验组(n=5),建立直径5 mm颅骨骨缺损模型,空白组未填充生物材料,对照组填充壳聚糖/羟基磷灰石支架,实验组填充柚皮苷-壳聚糖/羟基磷灰石复合支架。术后4周取材,CT扫描观察颅骨修复情况,苏木精-伊红染色观察颅骨修复的形态学,骨形态发生蛋白2和血管内皮生长因子免疫组化染色后观察缺损区域局部成骨活性因子的表达。结果与结论:(1)CT扫描显示,空白组大鼠颅骨未见明显骨生成,仅在缺损边缘可见少量新生骨;对照组于缺损孔隙可见新生骨形成,新生骨较少;实验组骨缺损修复良好,新生骨组织与缺损孔隙周围颅骨密度相似,大面积新生骨广泛填充了缺损孔隙。(2)苏木精-伊红染色显示,空白组缺损区填充以稀薄的疏松网状纤维组织,可见大量炎性反应病灶,...     

Comparison of bone regeneration in a rabbit skull defect by recombinant human BMP-2 incorporated in biodegradable hydrogel and in solution

The objective of this study is to compare bone regeneration induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) incorporated into a biodegradable gelatin hydrogel with that by rhBMP-2 in aqueous solution. After treating rabbit skull defects of 6 mm diameter with the two rhBMP-2 dosage forms, both of them increased the bone mineral density (BMD) at the skull defects with implantation time to a significantly higher extent than a rhBMP-2-free aqueous solution and a rhBMP-2-free empty gelatin hydrogel (p < 0.05). There was no quantifiable difference in BMD between the two dosage forms of rhBMP-2. Histological examination revealed that the integrity of newly generated bone increased with the rhBMP-2 dose, irrespective of the dosage form. The bone defect was filled with regenerated bone 21 days after treatment.     

负载rh-BMP2的羟基磷灰石人工骨治疗胫骨平台骨折骨缺损的临床疗效研究

目的 通过与羟基磷灰石生物陶瓷人工骨对比,评估负载rh-BMP2的羟基磷灰石人工骨对胫骨平台骨折骨缺损修复的临床疗效。方法 回顾性分析2014年10月至2019年9月中国人民解放军联勤保障部队第九二〇医院骨科收治的53例SchatzkerⅤ、Ⅵ型胫骨平台骨折骨缺损患者,所有患者均进行骨折切开复位内固定术并予术中植骨,其中27例患者术中予负载rh-BMP2的羟基磷灰石人工骨植骨（研究组）,另26例患者予羟基磷灰石生物陶瓷人工骨植骨（对照组）。对比两组患者骨折愈合时间、膝关节HSS功能评分、Rasmussen影像学评分及术后并发症发生情况。结果 所有患者获得随访,平均随访时间为（10.45±2.31）个月,所有患者最终均骨性愈合。研究组的骨折愈合时间[（13.46±3.38）周]明显短于对照组[（16.67±6.51）周]（P<0.05）;术后6个月时研究组HSS评分[（85.53±5.30）分]优于对照组[（80.58±6.40）分]（P<0.05）。术后6个月时两组的Rasmussen影像学评分比较,差异无统计学意义（P>0.05）,同时两组患者随访期间出现并发症情况...     

中空羟基磷灰石复合rhBMP-2修复骨缺损过程的再血管化研究

目的　探讨和观察中空羟基磷灰石复合rhBMP-2在骨缺损修复过程的再血管化。  方法　将48只成年的新西兰雄性大白兔制作成桡骨骨缺损模型,随机分3组,各组分别植入以下材料：中空HA/ rhBMP-2复合人工骨、单纯中空HA人工骨、单纯rhBMP-2。植入后于4、8、12、16周分别注射99mTc-MDP进行放射性核素骨显像并监测骨缺损修复过程中再血管化情况,同时进行大体、X线、组织学观察。  结果　术后各时间段,中空HA/ rhBMP-2复合人工骨组在X线及放射性核素聚集强度明显高于单纯中空HA人工骨组(P<0.05) ,表现为成骨代谢活跃及早期的再血管化能力。  结论　中空HA/ rhBMP-2复合人工骨具有良好的骨缺损修复能力,成骨活性持久,再血管化能力强,有望成为一种理想的骨缺损修复材料。     

核素骨显像评价组织工程骨修复骨缺损效果的价值

目的 应用放射性核素骨显像评价组织工程骨修复兔股骨髁骨缺损的效果。方法 取人白兔15只，抽取骨髓，行骨髓间充质干细胞分离、培养、骨向诱导。双侧股骨髁制作0．6×1．2cm的骨缺损，将诱导的成骨细胞复合珊瑚羟基磷灰石植入左侧，右侧单纯植入羟基磷灰石为对照组。术后4间、8周和12周分别行静态核素骨显像评价骨缺损的修复能力。结果表明术后4、8、12周实验组ROI计数（单位像素）均较对照组有显著性增高（P〈0．001）。实验组ROI计数随时间的延长呈明显的上升趋势，但术后8周始增长放缓；对照组ROI也有卜升趋势，但术后8周始增长加快，均在12周达到峰值。结论 骨髓间充质干细胞诱导后复合珊瑚羟基磷灰石可有效的修复股骨髁松质骨缺损。放射性核素骨显像在骨修复过程中具有动态评价血管化和骨生长的作用。     

Histological comparison of bone induced from autogenously grafted periosteum with bone induced from autogenously grafted bone marrow in the rat calvarial defect model

Both periosteum and bone marrow have the potential to induce heterotopic bone when grafted. Whether the process of bone formation is controlled by the recipient environment where the donor graft is placed or by factors from the donor site is not well documented. The purpose of this study was to examine the histology of new bone induced by either autogenously grafted periosteum or autogenously grafted bone marrow using the rat calvarial defect model in Sprague–Dawley rats. Grafts of either bone marrow or periosteum obtained from tibias were placed in calvarial defects with beta-tricalcium phosphate. Ten days after grafting, active cell proliferation was observed in the defects of both types of grafts. After 20 days, cancellous bone formation was observed in the defects with bone marrow grafts, and intramembranous bone formation was observed in the defects with periosteal grafts. After 30 days, bone marrow grafts had developed bone with a bone marrow-like structure, and the periosteal grafts had produced cortical bone structure in the defects. The findings suggest that the type of bone formation is determined by characteristics of the donor site.     

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(?-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.     

The effect of an AMPA antagonist (NBQX) on postischemic neuron loss and protein synthesis in the rat brain

Two groups of rats were subjected to 12 min of global cerebral ischemia and 6 days recirculation using the four-vessel occlusion model with hypotension and then treated with the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxa-linedione (Honoré et al. 1988). One group was used for routine and quantitative histology and immunostaining for glial fibrillary acidic protein (GFAP). The second group was subjected to autoradiographic studies of regional cerebral protein synthesis, with special emphasis on the hippocampus, the frontal cortex, and the thalamus. It was found that neuroprotective treatment with NBQX normalized cerebral protein synthesis rate (CPSR) in all investigated regions 6 days after ischemia. In untreated ischemic animals CPSR was normalized in all regions except for the CA3 and thalamus, where it had increased by 29% and 41%, respectively. Treatment of controls with NBQX had no effect on CPSR after 6 days. The histological investigations revealed that NBQX did not protect vulnerable cells in the dentate hilus and the reticular thalamic nucleus (RTN). In these regions reactive astrocytosis visualized by GFAP immunostaining was equally pronounced in both ischemic and NBQX-treated animals, and most neurons in the RTN were eosinophilic. The 80–100% pyramidal neuron loss in CA1 was accompanied by a high degree of reactive astrocytosis, whereas the NBQX-treated animals showed no signs of astrocytosis in this region. The ischemic CA1 pyramidal layer was also massively invaded by microglia. Together these observations strongly suggest that the quantitatively normal protein synthesis in this region after ischemia must be attributed to these glial cell populations.     

基因转染间充质干细胞修复大鼠骨缺损及其生物力学研究

目的评价腺病毒介导的人BMP-2基因转染的间充质干细胞对长骨骨干节段性骨缺损的修复效果。方法:36只雄性Wistar大鼠随机分为3组,每组12只,采用单侧股骨缺损模型,分别植入下列同系细胞与胶原的复合物:(1)Adv-hBMP-2转染的间充质干细胞;(2)Adv-βgal转染的间充质干细胞;(3)未转染细胞。于术后2、4、6、8周行放射学检查,术后8周的标本行组织学检查及生物力学测试。结果放射学检查显示,Adv-hBMP-2转染组术后2周有明显骨痂形成,术后8周12例骨缺损中有10例愈合,其他2组骨缺损均未愈合,放射学评分低于Adv-hBMP-2转染组,差异有统计学意义。组织学分析表明,Adv-hBMP-2转染组术后8周骨缺损内有丰富的编织骨和板层骨,其他2组无明显骨痂或仅有少量骨形成。生物力学测试显示,Adv-hBMP-2转染组手术侧股骨的弯曲强度是对侧未手术股骨的85%±8%。结论Adv-hBMP-2基因转染的同系大鼠间充质干细胞可修复长骨干节段性骨缺损。