Three-dimensional graphene oxide-coated polyurethane foams beneficial to myogenesis

Abstract

The development of three dimensional (3D) scaffolds for promoting and stimulating cell growth is one of the greatest concerns in biomedical and tissue engineering. In the present study, novel biomimetic 3D scaffolds composed of polyurethane (PU) foam and graphene oxide (GO) nanosheets were designed, and their potential as 3D scaffolds for skeletal tissue regeneration was explored. The GO-coated PU foams (GO-PU foams) were characterized by scanning electron microscopy and Raman spectroscopy. It was revealed that the 3D GO-PU foams consisted of an interconnected foam-like network structure with an approximate 300 μm pore size, and the GO was uniformly distributed in the PU foams. On the other hand, the myogenic stimulatory effects of GO on skeletal myoblasts were also investigated. Moreover, the cellular behaviors of the skeletal myoblasts within the 3D GO-PU foams were evaluated by immunofluorescence analysis. Our findings showed that GO can significantly promote spontaneous myogenic differentiation without any myogenic factors, and the 3D GO-PU foams can provide a suitable 3D microenvironment for cell growth. Furthermore, the 3D GO-PU foams stimulated spontaneous myogenic differentiation via the myogenic stimulatory effects of GO. Therefore, this study suggests that the 3D GO-PU foams are beneficial to myogenesis, and can be used as biomimetic 3D scaffolds for skeletal tissue engineering.     

The effects of co-delivery of BMSC-affinity peptide and rhTGF-β1 from coaxial electrospun scaffolds on chondrogenic differentiation

Electrospinning is a promising technology for the fabrication of scaffolds in cartilage tissue engineering. Two other important elements for tissue engineering are seed cells and bioactive factors. Bone marrow-derived stem cells (BMSCs) and rhTGF-β1 are extensively studied for cartilage regeneration. However, little is known about scaffolds that can both specifically enrich BMSCs and release rhTGF-β1 to promote chondrogenic differentiation of the incorporated BMSCs. In this study, we first fabricated coaxial electrospun fibers using a polyvinyl pyrrolidone/bovine serum albumin/rhTGF-β1 composite solution as the core fluid and poly(ε-caprolactone) solution as the sheath fluid. Structural analysis revealed that scaffold fibers were relatively uniform with a diameter of 674.4 ± 159.6 nm; the core–shell structure of coaxial fibers was homogeneous and proteins were evenly distributed in the core. Subsequently, the BMSC-specific affinity peptide E7 was conjugated to the coaxial electrospun fibers to develop a co-delivery system of rhTGF-β1 and E7. The results of ¹H nuclear magnetic resonance indicate that the conjugation between the E7 and scaffolds was covalent. The rhTGF-β1 incorporated in E7-modified scaffolds could maintain sustained release and bioactivity. Cell adhesion, spreading, and DNA content analyses indicate that the E7 promoted BMSC initial adhesion, and that the scaffolds containing both E7 and rhTGF-β1 (CBrhTE) were the most favorable for BMSC survival. Meanwhile, CBrhTE scaffolds could promote the chondrogenic differentiation ability of BMSCs. Overall, the CBrhTE scaffold could synchronously improve all three of the basic components required for cartilage tissue engineering in vitro, which paves the road for designing and building more efficient tissue scaffolds for cartilage repair.     

Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

The mitogen-activated protein kinase (MAPK) signaling cascade is an intracellular signaling pathway that is activated by diverse external stresses and regulates various cellular functions such as differentiation and proliferation (1). MAPK activation cascades consist of three components: MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. MAP3K phosphorylates and activates MAP2K, which in turn phosphorylates and activates MAPK (1, 2). In mammals, there are four distinct MAPK groups, ERKs (ERK1 and 2), JNKs (JNK1, 2, and 3), p38 (p38α through δ), and ERK5, each of which has its own upstream MAP3Ks and MAP2Ks (2, 3).A long-standing question in biochemistry is how diverse input signals can generate a specific MAPK signaling cascade. In other words, it is unknown how MAPK signaling modules dynamically interact in a spatiotemporal manner. To accomplish signaling fidelity, scaffolding proteins play an important role in colocalizing MAPK signaling modules. MAPK scaffolding proteins facilitate activation, regulate subcellular localization, and/or modulate the negative feedback of a specific MAPK signaling, which helps to organize a specific MAPK module to link the input signaling to proper biological outcomes (4, 5). Owing to the diverse roles of MAPK scaffolding proteins, they have been considered as potential therapeutic targets (6, 7).In mammals, a number of MAPK scaffolding proteins, including JIP, KSR, paxillin, MORG1, JSAP1, and arrestins, have been identified (8, 9). The functional role of MAPK scaffolding and the molecular and structural mechanisms of how the MAPK scaffolding proteins interact with MAPK signaling components vary depending on the scaffolding proteins (4). Therefore, it is important to study the details of the scaffolding mechanism of each MAPK scaffolding protein to gain a better understanding of the MAPK signaling mechanism and to precisely regulate a specific MAPK signaling pathway for therapeutic purposes.Arrestins were first discovered as proteins that play a key role in homologous desensitization and internalization of G protein–coupled receptors (GPCRs) (10). Four arrestins (arrestin-1 through -4) have been identified in humans: arrestin-1 and -4 (visual and cone arrestins, respectively) are expressed exclusively in the visual system, whereas arrestin-2 and -3 (also known as β-arrestin1 and 2, respectively) are ubiquitously expressed (10). Agonist-activated GPCRs, after coupling with G proteins, are phosphorylated by GPCR kinases. Arrestins bind active phosphorylated GPCRs, precluding receptor coupling to G proteins and facilitating receptor internalization (10, 11). In GPCR internalization, arrestins act as scaffolding proteins, linking the receptor with components of internalization machinery, such as clathrin and AP-2 (11–15). Arrestins have also been reported to interact with numerous signaling proteins, including MAPKs, and perform multiple functions (16, 17). The interaction with these signaling proteins occurs either in the basal or GPCR-induced active state of arrestins (11, 15, 18).Arrestins are the only known scaffolding proteins for MAPKs that are regulated by GPCRs. The interaction between MAPKs and arrestins has been extensively studied (11, 15, 19–21). Arrestins regulate GPCR-mediated ERK1/2 activation by scaffolding cRaf-1, MEK1, and ERK1/2 (22, 23) and regulate GPCR-dependent or -independent JNK3 signaling by scaffolding ASK1, MKK4/7, and JNK3 (24–26). Although the cellular and physiological effects of arrestins in MAPK signaling cascades have been studied extensively, the structural mechanisms governing arrestin/MAPK interactions have not been fully elucidated. Only a few studies have suggested a scaffolding mechanism of arrestins for ERK1/2 and JNK3 signaling components. A molecular simulation approach has suggested the binding interfaces of GPCR, arrestin-2, cSrc, cRaf, MEK1, and ERK1 (27), and mutation or truncation studies have investigated the interaction sites between arrestin and ERK1/2 signaling components (22, 28). Recent studies suggested an arrestin-3-mediated scaffolding and signal amplification mechanism of the JNK3 cascade (26, 29).Here, we studied the interaction of ERK1/2 signaling cascade components (cRaf, MEK1, and ERK2) with arrestin-2 using a combination of hydrogen/deuterium exchange–mass spectrometry (HDX-MS), Trp-induced bimane fluorescence quenching, and NMR spectroscopy. HDX-MS measures the exchange rate between the hydrogen atoms of amides in the protein backbone and the deuterium atoms in the solvent, which can provide an insight into protein–protein binding interfaces (30). NMR and Trp-induced bimane fluorescence quenching enable the detailed mapping of interaction interfaces within proteins (29, 31).     

蛛丝蛋白复合材料小直径血管支架的

探讨蛛丝蛋白复合材料小直径血管支架的体内降解性能和生物相容性,为其临床应用奠定基础。通过静电纺丝仪,将RGD 重组蛛丝蛋白(pNSR16)、聚己内酯(PCL)、明胶(Gt)、壳聚糖(CS)共混形成的纺丝液进行电纺,制得(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)支架,并将其植入SD大鼠腿部肌肉中,通过肉眼外观观察、组织切片HE染色评价等方法,评价蛛丝蛋白复合材料小直径血管支架的体内降解情况。分析支架浸提液对间充质干细胞集落形成、细胞分裂指数、台盼蓝拒染率、细胞毒性和细胞周期的影响,评价血管支架的生物相容性。血管支架在整个植入期内不断降解,(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)支架降解程度更深,纤维断裂严重,12周时失重率为20.3%,其降解速度明显快于(PCL/CS)/(PCL/Gt)支架,后者在12周时仅降解了13.2%。在(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)支架浸提液培养条件下的大鼠骨髓MSC集落生成率、平均集落面积和分裂指数都显著高于(PCL/CS)/(PCL/Gt)支架组。血管支架毒性等级均低于1 级,无细胞毒性。与血管支架浸提液复合培养的MSC生长状态良好,台盼蓝拒染率高于95%,复合培养48 h后,细胞G_0/1期比例降低,S、G₂/M期比例均升高。蛛丝蛋白复合材料小直径血管支架的体内降解和生物相容性良好,应用于临床具有一定的可行性。     

TGF-β1诱导分化的兔BMSCs复合左旋聚乳酸\β-磷酸三钙多孔支架材料体外研究实验

目的 研究体外培养rBMSCs经TGF-β1诱导分化的软骨细胞复合左旋聚乳酸\β-磷酸三钙(PLLA\β-TCP)多孔支架材料体外构建仿生人工软骨. 方法 低温挤出成形法制备成PLLA\β-TCP复合多孔支架材料,体外分离、培养rBMSCs至第3代,利用含有TGF-β1特殊诱导系统诱导其向软骨细胞分化,诱导14d后用甲苯胺蓝染色及Ⅱ型胶原免疫组化进行鉴定后与PLLA\β-TCP多孔支架材料体外复合培养,并取第7、14、21d细胞复合材料进行电镜扫描观察细胞贴附、生长、增殖状况,同时消化收集贴附支架第7、14、21d的细胞,行RT-PCR检测分化软骨细胞相关基因aggrecan、Co12A1在mRNA水平的表达,Western-bolt检测Ⅱ型胶原蛋白的分泌情况.结果 rBMSCs经诱导后向软骨细胞分化,甲苯胺蓝染色见分化软骨细胞分泌糖胺聚糖(glycosaminoglycan,GAG),Ⅱ型胶原免疫组织化学染色呈阳性;电镜扫描见分化细胞在支架材料分布均匀,黏附良好;RT-PCR及Westem-bolt检测示7、14、21daggrecan、Co12A1在mRNA水平、Ⅱ型胶原蛋白均有不同程度表达. 结论 利用含有TGF-β1特殊诱导系统诱导rBMSCs分化的软骨细胞复合到PLLA\β-TCP多孔支架材料上,细胞生长良好,并能正常分泌软骨细胞特异细胞外基质,体外成功构建了组织工程软骨.     

神经干细胞-施万细胞-聚乳酸-羟基乙酸共聚物支架移植对脊髓损伤大鼠的影响

目的探讨种植神经干细胞(NSCs)与施万细胞(SCs)的聚乳酸-羟基乙酸共聚物(PLGA)支架移植促进脊髓损伤大鼠神经功能恢复的作用及机制。方法体外培养NSCs 和SCs,以PLGA 为支架移植入大鼠T8 半横断脊髓损伤处。实验动物随机分为PLGA组、PLGA+NSCs 组和PLGA+NSCs+SCs 组。术前和术后进行皮层运动诱发电位(CMEPs)检查及BBB评分;然后在同侧或对侧进行T6再次半横断,并进行CMEPs 检测及BBB 评分。结果CMEPs 的恢复率及波幅在PLGA+NSCs+SCs 组最高。移植后,大鼠BBB 评分逐渐改善;在移植后第2 周及以后,PLGA+NSCs 组和PLGA+NSCs+SCs 组的BBB 评分显著高于PLGA 组(P<0.001)。同侧再次半横断后,CMEPs 消失, BBB 评分快速恢复;对侧再次半横断后,大鼠双下肢完全瘫痪。结论种植NSCs 和SCs 的PLGA支架移植有利于脊髓损伤功能重建,再生轴突可能形成了功能性连接;但是同侧再生轴突对脊髓功能的恢复作用有限。     

Esophagus and regenerative medicine

In addition to squamous cell carcinoma, the incidence of Barrett’s esophagus with high-grade dysplasia and esophageal adenocarcinoma is rapidly increasing worldwide. Unfortunately, the current standard of care for esophageal pathology involves resection of the affected tissue, sometimes involving radical esophagectomy. Without exception, these procedures are associated with a high morbidity, compromised quality of life, and unacceptable mortality rates. Regenerative medicine approaches to functional tissue replacement include the use of biological and synthetic scaffolds to promote tissue remodeling and growth. In the case of esophageal repair, extracellular matrix (ECM) scaffolds have proven to be effective for the reconstruction of small patch defects, anastomosis reinforcement, and the prevention of stricture formation after endomucosal resection (EMR). More so, esophageal cancer patients treated with ECM scaffolds have shown complete restoration of a normal, functional, and disease-free epithelium after EMR. These studies provide evidence that a regenerative medicine approach may enable aggressive resection of neoplastic tissue without the need for radical esophagectomy and its associated complications.     

细菌纤维素/明胶支架材料在成骨细胞体外培养中的应用

目的探索物理混合及化学交联两种方法制备的细菌纤维素(BC)/明胶支架材料用于成骨细胞体外培养的可行性。方法采取物理混合和化学交联两种方法将BC和明胶两者结合制备出海绵状复合体,接种成骨细胞后培养观察细胞形态,考察ALP活性表达,HE染色和共聚焦观察材料表面细胞情况和粘附。结果物理混合法制备的BC/明胶细胞增殖和粘附性能,ALP表达量优于单纯BC和化学交联制备的BC/明胶。结论物理混合法制备BC/明胶复合支架材料可以作为成骨细胞体外培养的良好载体。     

细胞片层技术的研究进展

组织工程的发展表现出了巨大的潜力,然而传统的组织工程方法限制了组织工程学的发展。新的细胞片层技术应用温度反应性培养皿收获细胞,避免了传统方法的不足之处。该方法不使用蛋白水解,仅简单地改变温度反应性培养皿的温度,便可收获完整的细胞片层。应用细胞片层进行组织重建的方式主要有将片层直接植入受区位点、通过同型或异型细胞片层的叠加构建三维结构的组织以及细胞片层结合支架的方法构建三维结构的组织。细胞片层技术与传统的组织工程学方法结合将会促进组织工程学的发展。