重组人骨形成蛋白-2的质量控制

目的建立重组人骨形成蛋白-2(rhBMP-2)质控方法和质量标准。方法以小鼠体内异位成骨试验测定rhBMP-2的生物学活性,HPLC-SEC法测定纯度,蛋白N-末端测序仪测定N-末端15个氨基酸残基序列,其余检测项目按《中国药典》三部(2005版)规定进行。结果1批rhBMP-2原液鉴定结果显示,样品比活性为5.9×104BU/mg,纯度为97.6%,N-末端序列为(MKR)LKSSCKRHPLYV,其余项目检测结果均符合《中国药典》三部(2005版)的要求。结论该质控标准可用于rhBMP-2产品的常规检定。     

间充质干细胞及基因转移方法促进新骨生成和大节段骨缺损的修复

目的构建含人骨形态蛋白-2(BMP-2)基因的重组腺病毒,并转染入间充质干细胞,诱导其向骨细胞分化,从而生成新骨并修复骨缺损。方法用重组腺病毒作为载体,将人BMP-2基因转染入小鼠胚胎间充质干细胞,Western blot检测BMP-2蛋白的分泌表达。观察重组腺病毒Adv-BMP-2转染后小鼠间充质干细胞的增殖变化,并进行碱性磷酸酶活性、钙化结节形成和细胞中成骨相关蛋白mRNA转录水平等细胞分化的指征检定。将Adv-BMP-2转染后的小鼠间充质干细胞注入裸鼠右侧大腿四头肌内,观察新骨生成情况。将重组腺病毒Adv-BMP-2转染的大鼠骨髓间充质干细胞用于大鼠大节段骨缺损的修复,并对植入的骨髓间充质干细胞进行跟踪观察。结果Western blot分析表明,重组腺病毒Adv-BMP-2转染的细胞可分泌表达BMP-2蛋白。转染后的小鼠间充质干细胞的增殖速度与重组腺病毒Adv-BMP-2的转染量呈剂量相关。Adv-BMP-2转染的干细胞碱性磷酸酶上升,并在体外形成钙结节,同时,成骨相关蛋白Osteopontin、Osteocalcin、Bone sialoprotein及Collagenα1(I)的mRNA水平也上升。用Adv-BMP-2转染的间充质干细胞能在裸鼠的大腿肌肉内形成发育成熟的新骨,转染的自体性骨髓间充质干细胞能有效修复大鼠大节段股骨缺损。在免疫抑制剂FK506的支持下,Adv-BMP-2转染的同种异体骨髓间充质干细胞也能修复大鼠的大节段骨缺损。没有FK506支持下的Adv-BMP-2转染的同种异体骨髓间充质干细胞及重组腺病毒Adv-β-gal转染的骨髓间充质干细胞则不能在体内形成新骨。植入骨缺损部位的骨髓间充质干细胞能直接参与骨缺损的修复,且有向全身其他组织器官迁移的趋势,但生存期较短。结论用腺病毒介导的BMP-2基因转染入间充质干细胞能有效诱导干细胞向骨细胞分化,生成新骨并修复骨缺损。     

Nanoscale Topographical Effects on the Adsorption Behavior of Bone Morphogenetic Protein-2 on Graphite

The interaction between bone morphogenetic protein-2 (BMP-2) and the surface of biomaterials is essential for the restoration of bone and cartilage tissue, inducing cellular differentiation and proliferation. The properties of the surface, including topology features, regulate the conformation and bioactivity of the protein. In this research, we investigated the influence of nanopatterned surfaces on the interaction of a homodimer BMP-2 with graphite material by combining molecular dynamics (MD) and steered molecular dynamics (SMD) simulations. The graphite substrates were patterned as flat, linear grating, square, and circular profiles in combination with BMP-2 conformation in the side-on configuration. Ramachandran plots for the wrist and knuckle epitopes indicated no steric hindrances and provided binding sites to type I and type II receptors. Results showed two optimal patterns that increased protein adsorption of the lower monomer while preserving the secondary structure and leaving the upper monomer free to interact with the cells. Charged residues arginine and lysine and polar residues histidine and tyrosine were the main residues responsible for the strong interaction with the graphite surface. This research provides new molecular-level insights to further understand the mechanisms underlying protein adsorption on nanoscale patterned substrates.     

重组人骨形态发生蛋白-7工程菌的高密度发酵

目的建立重组人骨形态发生蛋白-7(rhBMP-7)工程菌的高密度发酵工艺。方法采用摇瓶及发酵罐培养工程菌BL21/pBV221-rhBMP-7,观察不同培养基、乙酸浓度、pH值、诱导时间等对工程菌菌体生长及目的蛋白表达的影响。在优化的发酵条件下培养工程菌,当菌体A600值达100时,42℃升温诱导,并对表达产物进行纯化。结果发酵培养基与LB培养基培养的工程菌目的蛋白的表达量无明显差异;乙酸可明显抑制菌体生长及目的蛋白表达;最适于菌体生长和目的蛋白表达的pH值分别为6.8和7.6;最佳诱导时间为3h。以优化的发酵条件培养的工程菌诱导3h后,目的蛋白的表达量可达菌体总蛋白的34.9%,最终菌体A600值可达139.5;经纯化的目的蛋白纯度可达95%以上。结论已初步建立了rhBMP-7工程菌的高密度发酵工艺。     

重组人骨形态发生蛋白-7活性检测方法的建立

目的建立一种新的重组人骨形态发生蛋白-7(Recombinant human bone morphogenetic protein-7,rhBMP-7)活性检测方法。方法将吸收性明胶海绵与rhBMP-7混匀,冻干,环氧乙烷灭菌后,植入小鼠大腿股部肌间隙,共设5组:明胶海绵对照组及50、100、150、250μg rhBMP-7/块明胶海绵实验组,分别于术后第2、3周取材,进行HE染色及蛋白含量测定。结果 HE染色结果显示,与明胶海绵对照组相比,rhBMP-7/明胶海绵组细胞数量明显增多,且随着rhBMP-7剂量的增加及作用时间的延长而逐渐增多。蛋白含量测定结果显示,与明胶海绵对照组相比,rhBMP-7/明胶海绵组蛋白含量随着rhBMP-7剂量的增加及作用时间的延长而逐渐增加,且rhBMP-7剂量与每组蛋白含量平均值呈良好的相关性。结论将rhBMP-7复合明胶海绵植入小鼠大腿肌间隙并于第2周取材进行蛋白含量测定的方法,可用于rhBMP-7活性检测。     

Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.     

Stimulation of Bone Healing by Sustained Bone Morphogenetic Protein 2 (BMP-2) Delivery

The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (μCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p = 0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p = 0.006) with reduced cartilage at Day 84 (p = 0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p = 0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.     

重组人骨形态发生蛋白-7复合体对牙槽骨再生的影响

目的观察基因重组人骨形态发生蛋白-7(rhBMP-7)与明胶海绵的复合体对犬牙槽骨再生的影响。方法犬拔牙创内植入rhBMP-7与明胶海绵的复合体,以自然愈合拔牙创为对照。于术后2、4、8周分别将犬处死,取拔牙创处牙槽骨标本,行X线和组织学切片检查,观察成骨情况及牙槽骨萎缩情况。结果X线及组织学切片显示实验侧成骨速度及成骨质量明显优于对照侧。结论rhBMP-7与明胶海绵复合体是一种可促进牙槽骨再生的新型生物复合材料。     

Fabrication of Functionally Graded SiAlON Ceramics by Tape Casting

Functionally graded SiAlON ceramics (FG-SiAlONs) were successfully prepared by the tape casting and lamination approach. Non-aqueous SiAlON slurries with five different α to β-SiAlON ratios (85α:15β, 70α:30β, 55α:45β, 40α:60β, and 25α:75β) were prepared by a 66 methyl ethyl ketone/34 ethanol (vol%) mixture. Phase and microstructure analyses incorporation with hardness measurements clearly show that the FG-SiAlONs, prepared by tape casting, exhibit a continuous and gradual change in composition and hardness. Thus, the tape casting approach is a viable method to produce FG-SiAlONs with a precisely controlled composition, and subsequently properties, as a function of position.     

Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering.     

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein–substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.     

Spark Plasma Sintering of Functionally Graded SiO2/Mo Laminated Layer Material

A functionally graded sintered body consisting of laminated SiO₂/Mo layers was fabricated using spark plasma sintering. A high sintered‐body density was achieved using fine powders of SiO₂ (4.0 μm) and Mo (1.3 μm), which ensured no cracks were formed between layers. The highest joining stress (1.2 MPa) was found to occur at the interface between SiO₂ layers with 0 and 8 vol% Mo, with the electrical resistance decreasing rapidly when the Mo content was increased to 13 and 22 vol%. This is attributed to the use of spark plasma sintering, suggesting further work is needed to fully optimize this process.     

High-Intensity Discharge Lamp with Mo–SiO2 Functionally Graded Material

We show the invention of the new type of hermetically sealed high-intensity discharge lamps, made of Mo–SiO₂ functionally graded material (FGM) as an electrode and a sealing component. In the case of high-intensity discharge lamps with Mo–SiO₂ FGM (FGM lamp), the thermal expansion coefficient between Mo and SiO₂ is functionally graded so that it tolerates a large number of heating cycles, with no cooling system required. Furthermore, lamps survive without breakage. The W electrode is totally separated from the lamp envelope by the FGM, so that no leakage of the luminous elements or gases takes place, when a large gas pressure exists inside a lamp.     

Thermomechanical Analysis of Functionally Graded Thermal Barrier Coatings with Different Microstructural Scales

The recently developed two-dimensional version of the higher-order theory for functionally graded materials (denoted as HOTFGM-2D in previous communications) has been used to investigate the effects of microstructural architectures in graded thermal barrier coatings (TBCs) on stress distributions in the presence of a through-thickness temperature gradient. In particular, the response of TBCs with different levels of functionally graded microstructural refinement and different arrangements has been investigated, and the results for the through-thickness stress distributions are compared with those based on the standard micromechanical homogenization scheme. The examples presented illustrate the shortcomings of the standard micromechanics-based approach that is applied to the analysis of functionally graded TBCs, particularly if the presence of creep effects is included in the analysis.     

Mechanical Properties of Human Concentrated Growth Factor (CGF) Membrane and the CGF Graft with Bone Morphogenetic Protein-2 (BMP-2) onto Periosteum of the Skull of Nude Mice

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm³) or the CGF/BMP-2 (1.0 μg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery.     

颗粒共沉降方法消除梯度材料内部界面的可能性分析

本文旨在寻求一种结构控制手段,实现梯度材料在组成成份上连续分布.从颗粒共沉降的立场出发,建立了A/B系FGM沉降模型,并从理论上对用共沉降法制备A/B系FGM时,材料内部可能出现的界面情况进行了分析,提出了解决方案,为用共沉降法制备组成成份连续或准连续分布的FGM提供理论依据.     

Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

Anorganic bovine bone matrix (Bio-Oss^®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss^®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss^® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss^® or BMP-2/Bio-Oss^®. The Bio-Oss^® and BMP-2/Bio-Oss^® groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss^® group (16.50 ± 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss^® group (9.43 ± 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss^® markedly enhances bone regeneration.     

Calculation of the Composition Profile of a Functionally Graded Material Produced by Centrifugal Casting

Functionally graded materials have designed inhomogeneous distributions of different components on the scale of the material. They can be made by suspension processing, in which particles are stacked in a controlled manner. Segregation effects can be used to obtain the required gradient if the particles in suspension have different velocities. A model is derived for the composition profiles that develop as a result of these effects. The model can be used to determine the most suitable process conditions. The outcome of the model shows fair agreement with experiments found in the literature in which an Al₂O₃/ZrO₂ composite has been produced by uniaxial centrifugal casting.     

Concentration-Dependent Efficacy of Recombinant Human Bone Morphogenetic Protein-2 Using a HA/β-TCP Hydrogel Carrier in a Mini-Pig Vertebral Oblique Lateral Interbody Fusion Model

Bone morphogenetic protein-2 (BMP-2) is used in the treatment of degenerative spinal disease and vertebral fractures, spine fusion, dental surgery, and facial surgery. However, high doses are associated with side effects such as inflammation and osteophytes. In this study, we performed spinal fusion surgery on mini-pigs using BMP-2 and a HA/β-TCP hydrogel carrier, and evaluated the degree of fusion and osteophyte growth according to time and dosage. Increasing the dose of BMP-2 led to a significantly higher fusion rate than was observed in the control group, and there was no significant difference between the 8-week and 16-week samples. We also found that the HA + β-TCP hydrogel combination helped maintain the rate of BMP-2 release. In conclusion, the BMP-2-loaded HA/β-TCP hydrogel carrier used in this study overcame the drawback of potentially causing side effects when used at high concentrations by enabling the sustained release of BMP-2. This method is also highly efficient, since it provides mineral matter to accelerate the fusion rate of the spine and improve bone quality.