生物陶瓷骨种植及其与骨组织结合机理的探讨

一种新型的磷灰石类生物陶瓷锶磷灰石植入动物体内骨后，采用扫描电镜、能量散射电子探针和Ｘ线能谱等先进的分析学、组织学方法对植入材料与骨组织界面作早期观察，并探讨了该材料与骨组织结合的机理。实验结果表明锶磷灰石骨种植早期就发生Ｓｒ元素的迁移，它与骨结合的机理是由于早期的表面降解作用促进了骨重建。其研究结果提示了锶磷灰石陶瓷是具有良好生物相容性和表面活性的骨组织替代材料。     

三种新型植入材料的细胞毒性研究

羟基磷灰石及纯钛作为种植材料已广泛应用于临床,近年来,文献报道纯钛表面作氧化或氮化处理可提高其生物相容性,国内文献报告羟基磷灰石与骨水泥掺和使用可以增加羟基磷灰石的韧性,同时骨水泥可解决羟基磷灰石难以塑形的问题,本文通过细胞生长抑制试验和细胞毒性试验对羟基磷灰石——骨水泥复合材料,氧化钛,氮化钛三种材料的生物相容性作了初步评价,为临床应用提供生物学方面的依据.     

复合α-TCP透磷灰石骨水泥材料的生物相容性研究

目的:观察复合α-TCP透磷灰石骨水泥的生物相容性,为该复合骨水泥的临床应用提供动物组织学实验依据。方法:在β-TCP+MCPM骨水泥的基础上添加α-TCP,得到一种改进型透磷灰石骨水泥。以传统的透磷灰石骨水泥为对照组,对其进行体外溶血试验、热源试验、急性毒性试验、皮肤过敏试验、肌内植入试验。结果:α-TCP透磷灰石骨水泥的溶血率<5%,无热源性、无毒性、无皮肤过敏,植入肌肉后无明显炎症反应。结论:α-TCP的透磷灰石骨水泥具有良好的生物相容性和安全性,可作为体内骨替换材料。     

Interpore 500R复合人骨髓基质干细胞构建组织工程骨

目的：研究可吸收性珊瑚羟基磷灰石（Interpore 500R）作为骨组织工程支架材料的特点。方法：取人骨髓基质干细胞于含100ml/L胎牛血清的DMEM培养液中培养，并向成骨细胞诱导。将细胞与可吸收性珊瑚羟基磷灰石复合，通过扫描电镜观察细胞附着情况，共聚焦显微镜观察I型胶原的分泌。同时将细胞/Interpore500R复合物植入裸鼠体内构建骨组织。40d后取裸鼠标本组织切片、HE染色观察成骨情况。结果：骨髓基质干细胞在可吸收性羟基磷灰石表面附着良好，可分泌I型胶原，在裸鼠体内形成组织工程骨。结论：可吸收性羟基磷灰石生物相容性好，细胞附着后仍继续保持成骨细胞特性，并可形成骨组织。作为骨组织工程的支架材料，应用前景良好。     

骨生物衍生支架材料组织相容性实验研究

目的:研究骨生物衍生材料脱蛋白骨及脱钙骨的组织相容性,为进一步研究作为骨组织工程支架材料的可行性打下基础。方法:本实验应用急性毒性试验、亚急性毒性试验、血液相容性试验、肌肉刺激试验等,对骨生物衍生材料的组织相容性进行评价。结果:骨生物衍生材料(脱蛋白骨、脱钙骨)无急性亚急性毒性作用,具有良好的血液相容性,材料经肌肉埋植4周后,两种材料周围未见明显的炎症反应。结论:骨生物衍生材料(脱蛋白骨、脱钙骨)具有良好的生物相容性。     

纳米羟基磷灰石修复种植体周围骨缺损的实验研究

目的:探讨纳米羟基磷灰石材料在种植修复中的作用.方法:将纳米羟基磷灰石植入钛种植体周围的骨缺损区,观察种植体与骨界面的结合形式和程度.结果:纳米羟基磷灰石修复钛种植体骨缺损处实现良好的骨结合.结论:纳米羟基磷灰石材料的使用在实现钛种植体与周围健康骨组织的骨结合过程中起着重要的作用.     

牙种植术中几种骨量不足的处理

本文应用冻干骨粉、羟基磷灰石、可吸收骨诱导膜等植骨材料，和磁固位附着种植体处理牙种植术中牙槽骨骨量不足。通过对临床２７例患者的治疗，结果：１３例使用植骨材料，诱导骨组织再生，增加了牙槽骨宽度和高度，种植体植入后稳定，均健康存在。１４例患者采用磁性固位附着种植体，制作全口覆盖义齿。种植体生长良好，义齿的支持，固体，咀嚼功能恢复较好，取得了满意的效果。     

灌注羟基磷灰石的多孔NiTi合金牙种植材料的研究

目的检验灌注羟基磷灰石多孔NiTi合金牙种植材料的生物相容性。方法分别通过溶血试验、细胞毒性试验(MTT法),评价灌注羟基磷灰石多孔NiTi合金材料的生物安全性。结果灌注羟基磷灰石多孔NiTi合金材料无溶血现象,不影响凝血功能;MTT试验显示L929细胞在灌注羟基磷灰石多孔NiTi合金牙种植材料浸提液中生长良好,细胞毒性为0级。结论灌注羟基磷灰石多孔NiTi合金材料具有良好的生物安全性。     

水热合成法制备羟基磷灰石生物涂层的体内及体外实验研究

目的对水热合成法制备羟基磷灰石生物涂层材料的细胞相容性及动物体内植入体与骨界面的状况进行研究.方法水热合成法制备羟基磷灰石涂层,采用细胞培养和动物实验评估涂层材料的生物相容性.结果成骨细胞在涂层材料表面具有良好的细胞增殖率,碱性磷酸酶活性逐渐增加.植入动物体内1月后即有骨组织与涂层材料结合,3月后骨结合量增加.涂层材料在体内稳定,未见溶解脱落.结论水热合成法制备的羟基磷灰石生物涂层材料具有良好的生物效应.     

CGF 联合骨代用品及口腔修复膜在颌骨囊肿手术中的应用

目的：观察浓缩生长因子（ CGF）联合羟基磷灰石生物陶瓷及口腔修复膜在颌骨囊肿手术中促进骨组织愈合的临床效果。方法：45例颌骨囊肿患者行囊肿刮除术,采用浓缩生长因子及羟基磷灰石生物陶瓷混合物填充骨腔,CGF膜及口腔修复膜双层覆盖在骨缺损区表面。术后随访3～12个月,通过临床和影像学检查评估治疗效果。结果：45例患者术后伤口均为Ⅰ期愈合。直径＜2 cm的骨缺损在术后3个月羟基磷灰石生物陶瓷与周围骨组织界限消失,可见正常网纹结构的骨小梁。直径＞2 cm的骨缺损,术后9个月羟基磷灰石生物陶瓷与周围骨组织界限模糊;术后12个月界限消失,充填材料与新生骨及周围骨组织生长良好。结论：CGF联合羟基磷灰石生物陶瓷充填颌骨囊肿骨缺损区同时覆盖口腔修复膜的引导骨再生技术可有效促进骨愈合,修复骨缺损。     

人骨生物衍生材料细胞相容性的实验研究

目的:研究人骨生物衍生材料脱蛋白骨及脱钙骨的细胞相容性。方法:通过将人骨髓间充质干细胞(MSCs)与自行制备的骨生物衍生材料脱钙骨和脱蛋白骨在体外联合培养,了解骨生物衍生材料的细胞相容性。结果:骨髓MSCs可以在骨生物衍生材料表面黏附、增殖,材料对MSCs形态、增殖能力、ALP活性、细胞周期及倍体水平等均无明显不良影响。结论:骨生物衍生材料具有良好的细胞相容性,且脱蛋白骨优于脱钙骨。     

Methods to measure blood supply and detect the viability of the osteomyocutaneous flap

This article presents three kinds of atraumatic methods to detect the viability of revascularized bone grafts by microvascular anastomoses. Fluorescent assay during operation, 99mTc-MDP bone scan and semiconductor probe measurement and periodical pantomogram postoperatively were used. All the results were analysed quantitatively. The authors conclude that it is effective to detect the viability of the grafted bone with the integration of the three methods mentioned above.     

The role of RANKL and MMP‐9 in the bone resorption caused by ameloblastoma

J Oral Pathol Med (2010) 39 : 592–598 Background: Ameloblastoma, a common odontogenic tumor located in jaws, generally leads to severe damage to patient’s complexion and masticatory function. To expand in jaws, ameloblastoma must have a mechanism of resorbing the surrounding bone. Our objective was to explore the bone‐resorption mechanism of ameloblastoma by observing the role of Receptor activator of nuclear factor kappa B ligand (RANKL) and matrix metalloproteinase‐9 (MMP‐9) in the bone‐resorption process. Methods: In the study, the expression of RANKL and MMP‐9 in ameloblastoma was detected using immunohistochemistry (IHC) and RT‐PCR. Then, co‐culture system of ameloblastoma cells and bone marrow cells from neonatal rabbit was erected to observe the potential of ameloblastoma cells to induce osteoclastogenesis. Finally, the induced osteoclasts were used for in vitro bone‐resorption assay. In the co‐culture system and the bone‐resorption assay, the selective inhibitor of RANKL and MMP‐9, osteoprotegerin (OPG) and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) were, respectively, used for observing the role of RANKL and MMP‐9. Results: The expression of RANKL and MMP‐9 in ameloblastoma was confirmed. Ameloblastoma cells were found to induce bone marrow cells from neonatal rabbit differentiate into osteoclasts with bone‐resorption activity. In addition, OPG was found to, respectively, have markedly inhibitory effect on osteoclastogenesis (P < 0.01), and slightly inhibitory action on bone resorption (P < 0.05). Conclusions: Ameloblastoma cells had the potential to induce osteoclastogenesis. Moreover, RANKL played an essential role in the in vitro osteoclast formation and bone resorption induced by ameloblastoma cells.     

rhBMP-7对大鼠成骨细胞增殖及分化能力的影响

目的研究重组人骨形态发生蛋白-7（recombinant human bone morphogenetic proteins,rhBMP-7）对大鼠成骨细胞增殖和分化能力的影响。方法从SD大鼠取材进行原代培养,经碱性磷酸酶（alkaline phosphatase,ALP）和矿化结节检测鉴定为成骨细胞后传代至第3代,接种至96孔培养板。分为A、B、C、D、E、F共6组,每组3个复孔,rhBMP-7浓度分别为0．000、0．500、1．000、5．000、10．000、50．000mg/L。应用四甲基偶氮唑盐（methyl thiazolyl tetrazolium,MTT）法及PNPP偶氮法,分析不同浓度rhBMP-7作用72h后对大鼠成骨细胞增殖和ALP活性的影响,应用单因素方差分析和LSD法统计数据。结果作用72h后,6种浓度rhBMP-7对大鼠成骨细胞ALP活性促进作用的差异有统计学意义（F=11．840,P=0．000）,B组成骨细胞ALP活性高于A组,但差异无统计学意义（P=0．078）,C、D、E、F组大鼠成骨细胞ALP活性均高于A组（P值均为0．000）,rhBMP-7浓度为50．000mg／L时对ALP活性的影响最明显（P=0．000）。作用72h后6种浓度rhBMP-7对大鼠成骨细胞增殖影响的差异有统计学意义（F=2．726,P=0．026）;rhBMP-7能促进大鼠成骨细胞的增殖和分化,同A组（rhBMP-7浓度为0．000mg／L）相比,当rhBMP-7浓度为50．000mg／L时并作用至第3天时,大鼠成骨细胞增殖最显著（P=0．000）。结论一定剂量的rhBMP-7明显促进了大鼠成骨细胞的增殖和分化。     

Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study

Previous studies have shown that a combination of platelet-rich plasma (PRP) and autogenous bone graft can increase the rate of osteogenesis and enhance bone formation qualitatively. However, contradictory results were reported in a recent animal study. In order to clarify this inconsistency, this study examined the influence of the PRP concentrations on the viability and proliferation of alveolar bone cells in vitro. Bone cells obtained from the alveolar bone chips were exposed to various PRP concentrations. After a culture period of 7 days, cellular viability and proliferation were evaluated by counting the number of cells and a MTT assay. The results showed that the viability and proliferation of alveolar bone cells were suppressed by high PRP concentrations, but were stimulated by low PRP concentrations (1-5%). These in vitro results support the view that variations in the PRP concentrations might influence the bone formation within the PRP-treated bone grafts.     

A comparison of the alkaline phosphatases of rat dental pulp, bone, kidney, liver and intestine

The sensitivity of the dental pulp enzyme to heat was similar to that of enzymes from bone and kidney; alkaline phosphatases from liver and intestine were more stable to heat. L-Homoarginine strongly inhibited the enzyme activities from pulp, bone, kidney and liver but did not affect intestinal enzyme activity. Increasing the molarity of carbonate buffer or glycine buffer in the assay solution decreased intestinal alkaline phosphatase activity more markedly than enzyme activities of other tissues. In 100 mM glycine-NaOH buffer, the effects of Zn2+, Mg2+ and Ca2+ on pulp alkaline phosphatase activity were similar to those on the enzyme activities of bone, kidney and liver. The electrophoretic pattern of the pulp enzyme on sodium dodecyl sulphate-gels was identical with that of bone enzyme and differed from the patterns of enzymes from other tissues. These results suggest that the dental pulp alkaline phosphatase may be the same as bone enzyme.     

Comparison of the Capacity of Enamel Matrix Derivative Gel and Enamel Matrix Derivative in Liquid Formulation to Adsorb to Bone Grafting Materials

Background: The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). Methods: Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze‐dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold‐conjugated anti‐EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme‐linked immunosorbent assay (ELISA) kit for amelogenin. Results: The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate‐buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. Conclusions: The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.     

Effect of platelet releasate on bone cell migration and recruitment in vitro

The use of platelets and platelet products has become increasingly popular clinically as a means of accelerating endosseous wound healing. It is likely that growth factors released by activated platelets at the site of injury play a role in bone regeneration by stimulating the migration and proliferation of bone cells. In this study, a novel in vitro assay was developed to study the effects of platelet releasate (PR) collected from activated platelet concentrate on rat bone marrow-derived cells. Cultures of primary rat bone marrow cells were overlaid with a fibrin matrix, and the number of cells migrating within the three-dimensional matrix and the leading front of migration were quantified. The addition of PR to the top of the fibrin gels at different time points caused a 25% increase in the leading front of migration and a 3.5-fold increase in the number of migrating cells. Platelet releasate was also shown to have a mitogenic effect on bone cells in proliferation studies. Comparison between migration and proliferation data indicated that PR stimulates the initial recruitment of bone marrow cells to migration. This assay further allowed the determination that rat bone marrow cells are capable of exerting contractile forces on fibrin matrices and that matrix contraction is directly related to the migratory activity of cells. The results provide a potential mechanism to explain why biologically active platelet-derived factors enhance endosseous wound healing.     

兔骨髓间质干细胞诱导分化的生物特性研究

目的：本研究的目的在于研究骨髓间质干细胞体外诱导、分化培养的特性，以期寻找BMSC体外培养扩增和诱导分化的适宜时机和方法。方法：采用兔来源的骨髓间质干细胞体外扩增培养；选用不同代数的BMSC进行诱导分化，并行生物学检测。结果：兔骨髓间质干细胞可在体外培养扩增，形态类似成纤维样细胞；骨髓间质干细胞在条件培养液的存在下，可以分化成有成骨能力的细胞类型；体外可合成分泌类骨质钙盐。BMSC随代数的增加其生物反应性逐渐降低，并呈一定规律性。结论：骨髓间质干细胞可在体外传代扩增培养，在一定条件下可向成骨类型细胞转化，并随传代次数的增加，其转化效率与生物学性质成规律性变化。一定传代数的BMSC适于作为骨组织工程的种子细胞。     

同期神经化增强髂骨瓣术后骨髓间充质干细胞的活性

目的： 比较神经化髂骨瓣和传统髂骨瓣重建下颌骨缺损术后,移植骨块骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMMSCs）的干细胞活性。方法： 游离血管化髂骨瓣移植修复下颌骨缺损术后半年,从移植骨骨髓体外分离、培养BMMSCs,通过集落形成观察、Brdu摄入实验、群体倍增时间、体外成骨茜素红染色法、裸鼠皮下成骨法检测BMMSCs增殖、自我更新及成骨分化能力。采用SPSS 24.0软件包对数据进行统计学分析。结果： 神经化髂骨移植术后分离、培养的BMMSCs的集落形成、增殖、群体倍增时间、体外及体内成骨分化能力均显著高于非神经化组（P<0.05）。结论： 神经化髂骨瓣重建下颌骨缺损可以增强BMMSCs的自我更新、增殖、成骨分化等潜能,有助于维持移植骨的术后内环境稳定,减少术后移植骨吸收。