首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
《Acta biomaterialia》2014,10(2):883-892
Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans.  相似文献   

2.
Scaffolds for heart valve tissue engineering must function immediately after implantation but also need to tolerate cell infiltration and gradual remodeling. We hypothesized that moderately cross-linked collagen scaffolds would fulfill these requirements. To test our hypothesis, scaffolds prepared from decellularized porcine pericardium were treated with penta-galloyl glucose (PGG), a collagen-binding polyphenol, and tested for biodegradation, biaxial mechanical properties, and in vivo biocompatibility. For controls, we used un-cross-linked scaffolds and glutaraldehyde-treated scaffolds. Results confirmed complete pericardium decellularization and the ability of scaffolds to encourage fibroblast chemotaxis and to aid in creation of anatomically correct valve-shaped constructs. Glutaraldehyde cross-linking fully stabilized collagen but did not allow for tissue remodeling and calcified when implanted subdermally in rats. PGG-treated collagen was initially resistant to collagenase and then degraded gradually, indicating partial stabilization. Moreover, PGG-treated pericardium exhibited excellent biaxial mechanical properties, did not calcify in vivo, and supported infiltration by host fibroblasts and subsequent matrix remodeling. In conclusion, PGG-treated acellular pericardium is a promising scaffold for heart valve tissue engineering.  相似文献   

3.
The development of biomedical scaffolds mimicking a heterogeneous cellular microenvironment for a specified regulation of cell-fates is very promising for tissue engineering. In this study, three-dimensional scaffolds with heterogeneous microstructure were developed using a DMD-PP apparatus. During the fabrication process, this apparatus can efficiently switch monomers to form microstructures with localized, different material properties; the resolution in the arrangement of material properties is comparable to the characteristic size of functional subunits in living organs, namely, a hundred microns. The effectiveness of this DMD-PP apparatus is demonstrated by a woodpile microstructure with heterogeneous fluorescence and also by a microporous cell-culturing scaffold with selected sites for protein adhesion. Cell-cultivation experiment was performed with the microporous scaffold, in which selective cell adhesion was observed.  相似文献   

4.
This study examines the tissue engineering potential of type I collagen cross-linked in the presence of hydroxyapatite (HAp). Scaffolds were prepared by controlled freezing followed by lyophilization of composite mixtures of collagen and HAp in acetic acid, followed by cross-linking with 0.3% glutaraldehyde. Scaffolds of three ratios were prepared, corresponding to collagen/HAp ratios of 1:2, 1:4, and 1:6. The scaffolds were evaluated for their microstructure, chemical and physical properties, swelling behavior, mechanical strength, biodegradability hemocompatability, cytocompatibility, and histopathology following subcutaneous implantation in Sprague Dawley rats. The collagen/HAp matrices showed a smaller pore size of 10–40?μm compared to 50–100?μm for pure collagen scaffolds. Pure collagen showed a mechanical strength of 0.25?MPa, and the value almost doubled for cross-linked composites with collagen/HAp ratio 1:6. The improvement in mechanical strength corresponded to a decrease in swelling and enzymatic degradation (measured by resistance to collagenases). FTIR spectra results in conjunction with scanning electron micrographs showed that cross-linking in the presence of HAp did not significantly alter the structure of collagen. MTT assay and calcein AM staining revealed prominent and healthy growth of mesenchymal stem cells in both the pure collagen as well as collagen:HAp composites of ratio 1:2. In vivo implantation in Sprague Dawley rats showed an initial acute inflammatory response during days 3 and 7, followed by a chronic, macrophage-mediated inflammatory response on days 14 and 28. Overall, a cross-linked collagen/HAp composite scaffold of ratio 1:2 was identified as having potential for further development in tissue engineering.  相似文献   

5.
Marine biopolymer composite materials provide a technological platform for launching biomedical applications. Biomaterials demand good biocompatibility without the possibility of inflammation or foreign body reactions. In this study, we prepared two biocomposite hydrogels namely; HAC (hydroxyapatite, alginate & chitosan) and HACF (hydroxyapatite, alginate, chitosan & fucoidan) followed by calcium chloride cross linking. The prepared scaffolds were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Porosity measurement, swelling, biodegradation, hemolysis, RBC aggregation, plasma protein adsorption and cytotoxicity studies were also done. The hydrogel scaffold HACF possessed a well-defined porous architecture, sufficient water holding capacity, better hemocompatibility and biodegradability. The biocompatibility was confirmed through in vitro cytotoxicity studies such as MTT assay, Neutral red uptake, DAPI staining, Trypan blue dye exclusion test and direct contact assay in L929 mouse fibroblast cells. In addition, immunomodulatory and anti-inflammatory properties of both of these scaffolds were revealed by the mRNA expressions of major inflammatory marker genes in cytotoxic condition such as TNF-α, IL-6 and NF-κB. The physiochemical characterization and biological responses of HACF hydrogel signifies its suitability for various tissue engineering applications.  相似文献   

6.
目的 以聚乙烯醇(PVA)、壳聚糖(Cs)和胶原(Col)为主要原料制备PVA-Cs—Col复合支架,并研究其作为组织工程支架材料的可行性。方法 把聚乙烯醇、壳聚糖和胶原按一定配比复合,测定复合材料的含水率、膨胀率和力学性能,扫描电镜观察材料横截面的组织形态。结果 不同分子量PVA与不同质量的cs和Col复合,得到的复合支架材料湿态抗张强度为5.70MPa,含水率在60.15%-72.50%,膨胀率在185.33%~317.57%。不同配比的复合支架具有不同的内部组织形态结构。结论 PVA—Cs—Col复合支架材料具有较高的含水率和适宜的膨胀率,内部孔洞丰富,Cs:PVA:Col的质量比为30:15:0.20时,复合支架综合性能较佳,适合用于组织工程支架材料。  相似文献   

7.
Electrospinning has recently emerged as a leading technique for the formation of nanofibrous structures made of synthetic and natural extracellular matrix components. In this study, nanofibrous scaffolds were obtained by electrospinning a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type-I collagen in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HIFP). The resulting fibers ranged from 300 to 600 nm in diameter. Their surfaces were characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis and atomic force microscopy. The PHBV and collagen components of the PHBV/collagen nanofibrous scaffold were biodegraded by PHB depolymerase and a type-I collagenase aqueous solution, respectively. The cell culture experiments indicated that the PHBV/collagen nanofibrous scaffold accelerated the adhesion and growth of NIH3T3 cells more effectively than the PHBV nanofibrous scaffold, thus making the former a good scaffold for tissue engineering.  相似文献   

8.
Engineered tissues must utilize scaffolding biomaterials that support desired cellular functions and possess or can develop appropriate mechanical characteristics. This study assessed properties of collagen as a scaffolding biomaterial for ligament replacements. Mechanical properties of extruded bovine achilles tendon collagen fibers were significantly affected by fiber diameter, with smaller fibers displaying higher tangent moduli and peak stresses. Mechanical properties of 125 micrometer-diameter extruded fibers (tangent modulus of 359.6+/-28.4MPa; peak stress of 36.0+/-5.4MPa) were similar to properties reported for human ligaments. Scaffolds of extruded fibers did not exhibit viscoelastic creep properties similar to natural ligaments. Collagen fibers from rat tail tendon (a well-studied comparison material) displayed characteristic strain-softening behavior, and scaffolds of rat tail fibers demonstrated a non-intuitive relationship between tangent modulus and specimen length. Composite scaffolds (extruded collagen fibers cast within a gel of Type I rat tail tendon collagen) were maintained with and without fibroblasts under standard culture conditions for 25 days; cell-incorporated scaffolds displayed significantly higher tangent moduli and peak stresses than those without cells. Because tissue-engineered products must possess appropriate mechanical as well as biological/chemical properties, data from this study should help enable the development of improved tissue analogues.  相似文献   

9.
Novel porous aortic elastin and collagen scaffolds for tissue engineering   总被引:15,自引:0,他引:15  
Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.  相似文献   

10.
目的观察MC3T3-El成骨前体细胞在壳聚糖-脱细胞真皮三维支架材料上的黏附情况,并评价其细胞相容性。方法通过冷冻干燥制备壳聚糖-脱细胞真皮三维支架材料,并测试其孔隙率、密度和吸水率,通过扫描电镜分析支架的微观形貌。采用体外培养细胞的方法,将MC3T3-E1细胞直接接种到壳聚糖-脱细胞真皮三维支架材料上,培养2,3,4,5h,各时间点各取3个样品,测定细胞在支架上的黏附率,确定最佳的细胞贴壁时间。将细胞接种到支架上,共培养1,3,5,7,9,11,13d,采用MTS方法绘制细胞增殖曲线,组织化学染色观察细胞形态,并利用材料试验机测试不同时间材料细胞复合物的压缩弹性模量。结果壳聚糖-脱细胞真皮材料具有连通的多孔结构,孔隙率为92.8%,密度为97.96g/L,吸水率为(2169±100)%。细胞相容性实验显示,成骨细胞易于在支架材料上黏附、增殖。结论壳聚糖-脱细胞真皮材料具有连通的孔隙,孔径较均匀,MC3T3-El成骨前体细胞易在壳聚糖-脱细胞真皮三维支架材料上黏附、增殖,表明该支架材料具有良好的细胞相容性。  相似文献   

11.
A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(ε-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 μm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 μm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young’s modulus ~40 MPa and strength ~1 MPa) in comparison with the controls, despite the large porosity (~90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.  相似文献   

12.
In this study, a highly porous collagen-based biodegradable scaffold was developed as an alternative to synthetic, non-degradable corneal implants. The developed method involved lyophilization and subsequent stabilization through N-ethyl-N'-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking to yield longer lasting, porous scaffolds with a thickness similar to that of native cornea (500 microm). For collagen-based scaffolds, cross-linking is essential; however, it has direct effects on physical characteristics crucial for optimum cell behavior. Hence, the effect of cross-linking was studied by examining the influence of cross-linking on pore size distribution, bulk porosity and average pore size. After seeding the foam with human corneal keratocytes, cell proliferation, cell penetration into the scaffold and ECM production within the scaffold were studied. After a month of culture microscopical and immunohistochemical examinations showed that the foam structure did not undergo any significant loss of integrity, and the human corneal keratocytes populated the scaffold with cells migrating both longitudinally and laterally, and secreted some of the main constituents of the corneal ECM, namely collagen types I, V and VI. The foams had a layer of lower porosity (skin layer) both at the top and the bottom. Foams had an optimal porosity (93.6%), average pore size (67.7 microm), and chemistry for cell attachment and proliferation. They also had a sufficiently rapid degradation rate (73.6+/-1.1% in 4 weeks) and could be produced at a thickness close to that of the natural corneal stroma. Cells were seeded at the top surface of the foams and their numbers there was higher than the rest, basically due to the presence of the skin layer. This is considered to be an advantage when epithelial cells need to be seeded for the construction of hemi or full thickness cornea.  相似文献   

13.
In this study, a highly porous collagen-based biodegradable scaffold was developed as an alternative to synthetic, non-degradable corneal implants. The developed method involved lyophilization and subsequent stabilization through N-ethyl-N′-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking to yield longer lasting, porous scaffolds with a thickness similar to that of native cornea (500 μm). For collagen-based scaffolds, cross-linking is essential; however, it has direct effects on physical characteristics crucial for optimum cell behavior. Hence, the effect of cross-linking was studied by examining the influence of cross-linking on pore size distribution, bulk porosity and average pore size. After seeding the foam with human corneal keratocytes, cell proliferation, cell penetration into the scaffold and ECM production within the scaffold were studied. After a month of culture microscopical and immunohistochemical examinations showed that the foam structure did not undergo any significant loss of integrity, and the human corneal keratocytes populated the scaffold with cells migrating both longitudinally and laterally, and secreted some of the main constituents of the corneal ECM, namely collagen types I, V and VI. The foams had a layer of lower porosity (skin layer) both at the top and the bottom. Foams had an optimal porosity (93.6%), average pore size (67.7 μm), and chemistry for cell attachment and proliferation. They also had a sufficiently rapid degradation rate (73.6 ± 1.1% in 4 weeks) and could be produced at a thickness close to that of the natural corneal stroma. Cells were seeded at the top surface of the foams and their numbers there was higher than the rest, basically due to the presence of the skin layer. This is considered to be an advantage when epithelial cells need to be seeded for the construction of hemi or full thickness cornea.  相似文献   

14.
When traumatic injury, tumor removal, or disease results in significant bone loss, reconstructive surgery is required. Bone grafts are used in orthopedic reconstructive procedures to provide mechanical support and promote bone regeneration. In this study, we applied a heat sintering technique to fabricate 3D electrospun scaffolds that were used to evaluate effects of mineralization and fiber orientation on scaffold strength. We electrospun PLLA/gelatin scaffolds with a layer of PDLA and heat sintered them into three-dimensional cylindrical scaffolds. Scaffolds were mineralized by incubation in 10× simulated body fluid for 6, 24, and 48 h to evaluate the effect of mineralization on scaffolds compressive mechanical properties. The effects of heat sintering hydroxyapatite (HA) microparticles directly to the scaffolds on mineral deposition, distribution and mechanical properties of the scaffolds were also evaluated. We found that orientation of the fibers had little effect on the compressive mechanical properties of the scaffolds. However, increasing the mineralization times resulted in an increase in compressive mechanical properties. Also, the direct addition of HA microparticles had no effect on the scaffold mechanical properties, but had a significant effect on the mineral deposition on PLLA/gelatin scaffolds.  相似文献   

15.
Li Y  Ma T  Yang ST  Kniss DA 《Biomaterials》2001,22(6):609-618
Nonwoven fibrous matrices have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The method of thermal compression of nonwoven polyethylene terephthalate (PET) fabrics was developed and key parameters of temperature, pressure, and compression duration were evaluated in this study. The permanent deformation was obtained at elevated temperature under pressure and the viscoelastic compressional behaviors were observed, characterized by a distinct apparent modulus change in glass transition temperature region. A liquid extrusion method was further employed to analyze both pore size and its distribution for matrices with porosity ranging from 84 to 93%. It is also found that a more uniformly distributed pore size was resulted from thermal compression and the isotropic nature of nonwoven fabrics was preserved because of the proportional reduction of the pore by compression. The thermally compressed fabric matrices with two different pore sizes (15 and 20 microm in pore radius) were used to culture human trophoblast ED27 and NIH 3T3 cells. It was found that cells cultured in the different pore-size PET matrices had different cell spatial organization and proliferation rates. The smaller pores in the matrix allowed cells to spread better and proliferate faster, while cells in the larger pores tended to form large aggregates and had lower proliferation rate. The thermal compression technique also can be applied to other synthetic fibrous matrices including biodegradable polymers used in tissue engineering to modify the microstructure according to their viscoelastic properties.  相似文献   

16.
We present an electrospun synthetic human elastin:collagen composite scaffold aimed at dermal tissue engineering. The panel of electrospun human tropoelastin and ovine type I collagen blends comprised 80% tropoelastin+20% collagen, 60% tropoelastin+40% collagen and 50% tropoelastin+50% collagen. Electrospinning efficiency decreased with increasing collagen content under the conditions used. Physical and mechanical characterization encompassed fiber morphology, porosity, pore size and modulus, which were prioritized to identify the optimal candidate for dermal tissue regeneration. Scaffolds containing 80% tropoelastin and 20% collagen (80T20C) were selected on this basis for further cell interaction and animal implantation studies. 80T20C enhanced proliferation and migration rates of dermal fibroblasts in vitro and were well tolerated in a mouse subcutaneous implantation study where they persisted over 6weeks. The 80T20C scaffolds supported fibroblast infiltration, de novo collagen deposition and new capillary formation.  相似文献   

17.
This study investigates the potential of high density type I collagen gels as an injectable scaffold for tissue engineering of whole menisci, and compares these results with previous strategies using alginate as an injectable scaffold. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into micro-computed tomography-based molds to create 10 and 20 mg ml?1 menisci that were cultured for up to 4 weeks and compared with cultured alginate menisci. Contraction, histological, confocal microscopy, biochemical and mechanical analysis were performed to determine tissue development. After 4 weeks culture, collagen menisci had preserved their shape and significantly improved their biochemical and mechanical properties. Both 10 and 20 mg ml?1 menisci maintained their DNA content while significantly improving the glycosaminoglycan and collagen content, at values significantly higher than the alginate controls. Collagen menisci matched the alginate control in terms of the equilibrium modulus, and developed a 3- to 6-fold higher tensile modulus than alginate by 4 weeks. Further fibrochondrocytes were able to reorganize the collagen gels into a more fibrous appearance similar to native menisci.  相似文献   

18.
Extended soft tissue defects after extensive deep burns or tumor resections are still an unresolved problem in plastic and reconstructive surgery. There is a clinical need for an adequate solution to this problem but currently, no adequate implant material is available for the correction of these defects. Since the autologous transplantation of mature adipose tissue gives poor results, this study explores the advantages of using human preadipocytes in collagen sponges for tissue reconstruction purposes. Human preadipocytes of young adults were isolated, cultured, seeded onto collagen sponges with uniform pore size, and implanted into immunodeficient mice. After 24 hours of incubation in vitro and after explantation at 3, 8, and 12 weeks, sponges were examined for macroscopic appearance, weight, thickness, histology, immuno-histochemistry, and ultrastructure. We find good penetration of cells into the scaffold, layers of adipose tissue, and new vessels on all grafts while controls appear unchanged. These results are promising for improving the reconstruction of soft tissue defects.  相似文献   

19.
背景:可吸收天然胶原支架材料是成熟和理想的半月板替代物。 目的:总结半月板组织工程学研究的现状。 方法:以“组织工程学、运动性半月板损伤、种子细胞、可吸收天然胶原、生物支架材料、应力刺激、力学因素”为中文关键词,以“Tissue engineering、Movement of the meniscus、Seed cells、Natural collagen can be absorbed、Biological scaffolds、Stress stimulation、Mechanical factor”为英文关键词,采用计算机检索PubMed数据库和维普数据库中1994年1月至2011年12月与运动性半月板损伤及半月板组织工程研究相关的文章。 结果与结论:目前的研究重点包括半月板损伤机制、可吸收天然胶原作为半月板组织工程支架的可行性分析、应力刺激、半月板恢复力学因素4个方面。研究表明半月板组织工程修复运动性半月板损伤具有良好的应用前景和广阔的使用空间,但在实际应用中,半月板组织工程支架的构建、细胞外基质复合材料的研究及其与组织的相容性,修复后组织工程半月板的应力刺激和所能承受的力学因素问题仍是半月板组织工程学方面的难点问题。  相似文献   

20.
于强  田京 《中国组织工程研究》2015,19(30):4870-4875
背景:3D打印技术自20世纪末出现以来逐渐应用在医学领域已成为一种趋势。近年来3D打印技术被广泛用于骨组织工程支架材料的成型,并取得了一些令人惊喜的成果。 目的:文章从骨组织工程支架基本概念、3D打印的基本原理和流程、3D打印应用于构造支架的要求以及不同的粉末材料等方面进行阐述,分析其优势与目前存在的局限性,并对未来3D打印在骨组织工程支架中的应用进行展望。 方法:第一作者应用计算机检索1990年1月至2015年2月MEDLINE数据库、Science Direct全文数据库、中国期刊全文数据库、维普中文期刊网等有关3D打印技术在构建骨组织工程支架中应用的文章,检索词“3D打印,组织工程学,快速成型技术,支架,材料”,排除重复性研究。文章共检索到52篇相关文献,其中33篇文献符合纳入标准。 结果与结论:3D打印技术具有高精度、构建速度快、可按需制造实现个性化定制等优势。3D打印应用于骨组织工程支架构建时,所用的粉末或黏合剂需具备一定的条件,如流动性、稳定性与可湿性等。用于打印的粉末材料可分为人工合成多聚体、天然高分子聚合物、生物陶瓷及它们的混合物。不同粉末材料的粉末各自优缺点不同,且最终成型效果也不尽相同。3D打印技术也存在一些包括费用昂贵、不易大规模生产等方面的局限性。但尽管如此,3D打印的临床应用前景一片光明。 中国组织工程研究杂志出版内容重点:生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号