Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique

Automated manufacturing is a key for tissue-engineered therapeutic products to become common-place and economical. Here, we developed an automatic cell sheet stacking apparatus to fabricate 3-dimensional tissue-engineered constructs exploiting our cell sheet manipulator technique, where cell sheets harvested from temperature-responsive culture dishes are stacked into a multilayered cell sheet. By optimizing the stacking conditions and cell seeding conditions, the apparatus was eventually capable of reproducibly making five-layer human skeletal muscle myoblast (HSMM) sheets with a thickness of approximately 70–80 μm within 100 min. Histological sections and confocal topographies of the five-layer HSMM sheets revealed a stratified structure with no delamination. In cell counts using trypsinization, the live cell numbers in one-, three- and five-layer HSMM sheets were equivalent to the seeded cell numbers at 1 h after the stacking processes; however, after subsequent 5-day static cultures, the live cell numbers of the five-layered HSMM sheets decreased slightly, while one- and three-layer HSMM sheets maintained their live cell numbers. This suggests that there are thickness limitations in maintaining tissues in a static culture. We concluded that by combining our cell sheet manipulator technique and industrial robot technology we can create a secure, cost-effective manufacturing system able to produce tissue-engineered products from cell sheets.     

Gelatin-encapsulated iron oxide nanoparticles for platinum (IV) prodrug delivery,enzyme-stimulated release and MRI

A facile method for transferring hydrophobic iron oxide nanoparticles (IONPs) from chloroform to aqueous solution via encapsulation of FITC-modified gelatin based on the hydrophobic–hydrophobic interaction is described in this report. Due to the existence of large amount of active groups such as amine groups in gelatin, the fluorescent labeling molecules of fluorescein isothiocyanate (FITC) and platinum (IV) prodrug functionalized with carboxylic groups can be conveniently conjugated on the IONPs. The nanoparticles carrying Pt(IV) prodrug exhibit good anticancer activities when the Pt(IV) complexes are reduced to Pt(II) in the intracellular environment, while the pure Pt(IV) prodrug only presents lower cytotoxicity on cancer cells. Meanwhile, fluorescence of FITC on the surface of nanoparticles was completely quenched due to the possible Förster Resonance Energy Transfer (FRET) mechanism and showed a fluorescence recovery after gelatin release and detachment from IONPs. Therefore FITC as a fluorescence probe can be used for identification, tracking and monitoring the drug release. In addition, adding pancreatic enzyme can effectively promote the gelatin release from IONPs owing to the degradation of gelatin. Noticeable darkening in magnetic resonance image (MRI) was observed at the tumor site after in situ injection of nanoparticles, indicating the IONPs-enhanced T₂-weighted imaging. Our results suggest that the gelatin encapsulated Fe₃O₄ nanoparticles have potential applications in multi-functional drug delivery system for disease therapy, MR imaging and fluorescence sensor.     

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin,hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin–methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in Gel-MA-based hydrogels, and show that with the incorporation of small quantities of photocrosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesized extracellular matrix (ECM) throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 114 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.     

Therapeutic efficacy of antibiotic-loaded gelatin microsphere/silk fibroin scaffolds in infected full-thickness burns

Despite advances in burn treatment, burn infection remains a major cause of morbidity and mortality. In this study, an antibacterial silk fibroin (SF) scaffold for burn treatment was designed; gelatin microspheres (GMs) were impregnated with the antibiotic gentamycin sulfate (GS), and the GS-impregnated GMs were then embedded in a SF matrix to fabricate GS/GM/SF scaffolds. The developed GS/GM/SF scaffolds could serve as a dermal regeneration template in full-thickness burns. The average pore size and porosity of the GS/GM/SF scaffolds were 40–80 μm and 85%, respectively. Furthermore, the drug release rate of the scaffolds was significantly slower than that of either GS/GM or GS/SF scaffolds. And the composite scaffold exhibited stronger antimicrobial activities against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Hence, we evaluated the wound-healing effects and antibacterial properties of the GS/GM/SF scaffolds in a rat full-thickness burn infection model. Over 21 days, the GS/GM/SF scaffolds not only significantly reduced burn infection by P. aeruginosa but also accelerated the regeneration of the dermis and exhibited higher epithelialization rates than did GS/SF and SF scaffolds. Thus, GS/GM/SF scaffolds are potentially effective for treatment of full-thickness infected burns, and GS/GM/SF scaffolds are a promising therapeutic tool for severely burned patients.     

尼莫地平明胶微球的制备及其性质研究

目的研究尼莫地平明胶微球的制备工艺,并考察其体外释药特性。方法以天然的可生物降解的明胶为载体,液体石蜡为油相,Span80为乳化剂,采用正交设计优化空白明胶微球的制备工艺,用乳化法制备尼莫地平明胶微球。结果优选所制尼莫地平明胶微球形态圆整,大小均匀,表面光滑,载药量为13.48%。体外释药结果表明,一级动力学方程能较好地对其进行拟合。结论制备工艺稳定可行,所得尼莫地平明胶微球具有良好的缓释效果。     

胎兔成骨细胞与bFGF-明胶海绵复合物的体外共建

目的探讨bFGF-明胶海绵复合物作为骨组织工程支架的可行性,为进一步的体内实验提供基础研究。方法取第二代培养的成骨细胞分别与bFGF-明胶海绵复合物及明胶海绵复合培养,进行一般与超微形态学观察,观察细胞生长情况并做成骨细胞碱性磷酸酶(ALP)及微量蛋白含量的测定,比较细胞的粘附能力、增殖活力及成骨活性。结果bFGF-明胶海绵复合物、明胶海绵均可与成骨细胞复合生长,前者的细胞粘附能力、增殖活力及成骨活性均明显优于后者。结论bFGF-明胶海绵复合物能较好的发挥bF- GF作为信号因子的作用,并能提高明胶海绵的相容性,是构建组织工程骨的一种理想的支架材材。     

胶质瘤中MMPs酶活性和肿瘤分级相关性的研究

目的 探讨基质金属蛋白酶2(MMP-2)与基质金属蛋白酶9(MMP-9)的活性与胶质瘤临床分级之间的关系.方法 采用明胶酶谱50例人脑胶质瘤与4例正常脑组织中的MMP-2及MMP-9进行检测.结果 在各级别组中,MMP-2和MMP-9的酶原形式无差别;随着胶质瘤恶性程度的增加,MMP-2和MMP-9的活化酶含量显著增加.结论 在不同恶性级别的人脑胶质瘤中,MMP-2和MMP-9酶活性发生改变,这些改变具有显著临床意义.     

Acceleration of bone formation with BMP2 in frame-reinforced carbonate apatite–collagen sponge scaffolds

The development is expected of scaffold biomaterials that feature a shape-maintaining property in addition to high porosity and large pores that cells can easily invade. To develop a new biodegradable scaffold biomaterial reinforced with a frame, synthesized carbonate apatite (CO₃Ap) was mixed with neutralized collagen gel, and the CO₃Ap–collagen mixtures were lyophilized into sponges in a porous hydroxyapatite (HAp) frame ring. X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) analyses together with chemical analysis indicated that the synthesized CO₃Ap had a crystalline nature and a chemical composition similar to that of bone. Scanning electron microscope (SEM) observation showed that the CO₃Ap–collagen sponge had a sui pore size for cell invasion. In proliferation and differentiation experiments with osteoblasts, alkaline phosphatase and osteopontin activity were clearly detected. When these sponge–frame complexes with bone morphogenic protein (rh-BMP2) were implanted beneath the periosteum cranii of rats, significant new bone was created at the surface of the periosteum cranii after 4 weeks of implantation. These reinforced CO₃Ap–collagen sponges with rh-BMP2 are expected to be used as hard tissue scaffold biomaterials for the therapeutic purpose of the rapid cure of bone defects.     

埋伏阻生齿拔除术后两种拔牙窝填塞剂对预后的影响

目的 观察骨埋伏阻生牙拔除术后两种不同的牙槽窝填塞剂对预后的影响。方法 选取315例骨埋伏的下颌第三磨牙拔除的患者,随机分成3组,组1拔除术后创面直接拉拢缝合+口服镇痛+口服抗炎;组2拔除术后创面明胶海绵+碘仿粉末填塞止血+拉拢缝合+口服镇痛+口服抗炎;组3拔除术后创面胶质银明胶海绵(止泰)填塞止血+拉拢缝合+口服镇痛+口服抗炎。比较3组术后出血,术后肿痛及术后干槽症的发生率。结果 组2和组3创面处理治疗后的术后出血,术后肿痛及术后干槽症的出现明显降低,预后优于组1(P<0.01),且组2和组3两组之间差异无统计学意义(P>0.05)。结论 胶原蛋白海绵+碘仿粉末或胶质银明胶海绵的两种术后创面处理方法皆疗效显著,值得临床进一步推广和应用。     

饥饿期粪肠球菌生物膜毒力因子明胶酶E表达研究

目的:研究粪肠球菌生物膜形成相关毒力因子明胶酶E(gelE)在饥饿期及药物作用后的表达情况。方法:体外建立对数期、稳定期、饥饿期粪肠球菌生物膜模型,分别以1%、2.5%、5.25%次氯酸钠溶液作用于各时期粪肠球菌生物膜后,用Real-time PCR对gelE的基因表达相对量进行检测。结果:gelE基因表达相对量,饥饿期高于稳定期及对数期(P<0.05);用药后,3个期gelE基因表达相对量,5.25%次氯酸钠溶液组低于2.5%次氯酸钠溶液组和1%次氯酸钠溶液组(P<0.05)。结论:饥饿期粪肠球菌生物膜形成相关毒力因子gelE较对数期及稳定期表达增强。次氯酸钠浓度依赖性可抑制粪肠球菌毒力因子gelE的表达。