The effects of washing a collagen sample prior to TEM examination

Transmission electron microscopy (TEM) is an important analysis technique to visualize (bio)macromolecules and their assemblies, including collagen fibers. Many protocols for TEM sample preparation of collagen involve one or more washing steps to remove excess salts from the dispersion that could hamper analysis when dried on a TEM grid. Such protocols are not standardized and washing times as well as washing solvents vary from procedure to procedure, with each research group typically having their own protocol. Here, we investigate the influence of washing with water, ethanol, but also methanol and 2-propanol, for both mineralized and unmineralized collagen samples via a protocol based on centrifugation. Washing with water maintains the hydrated collagen structure and the characteristic banding pattern can be clearly observed. Conversely, washing with ethanol results in dehydration of the fibrils, often leading to aggregation of the fibers and a less obvious banding pattern, already within 1 min of ethanol exposure. As we show, this process is fully reversible. Similar observations were made for methanol and propanol. Based on these results, a standardized washing protocol for collagenous samples is proposed.     

海蜇Ⅰ型胶原蛋白的提取及结构特性研究

以海蜇为原料，采用盐酸-胃蛋白酶法提取胶原蛋白，并通过聚丙烯酰胺凝胶电泳（SDS-Polyacrylamide gel electrophoresis，SDS-PAGE）、紫外光谱（UV-visible spectroscopy，UV）、氨基酸分析、红外光谱（Fourier-transform infrared spectroscopy，FTIR）、圆二色谱（Circular dichroism，CD）以及扫描电镜（Scanning electron microscope，SEM）对其结构特性进行了系统全面的研究。结果显示，海蜇胶原蛋白在135 kDa左右有一条α-链，在245 kDa以上有一条β-链和γ-链，紫外吸收峰在233 nm处，符合I型胶原蛋白的特征，其亚基组成可能为[α₁(I)]₃。海蜇胶原蛋白中含量最高的氨基酸为甘氨酸，占氨基酸总量的25.99%，且含有15.94%的亚氨基酸。红外光谱和圆二色谱均表明海蜇胶原蛋白分子排布紧凑，具有主要由氢键构成的完整三螺旋结构。扫描电镜结果显示海蜇胶原蛋白具有多层聚集、以纤维为主的无规则网状结构。     

Magnetic Assembly of a Multifunctional Guidance Conduit for Peripheral Nerve Repair

Nerve growth conduits are designed to support and promote axon regeneration following nerve injuries. Multifunctionalized conduits with combined physical and chemical cues, are a promising avenue aimed at overcoming current therapeutic barriers. However, the efficacious assembly of conduits that promote neuronal growth remains a challenge. Here, a biomimetic regenerative gel is developed, that integrates physical and chemical cues in a biocompatible “one pot reaction” strategy. The collagen gel is enriched with magnetic nanoparticles coated with nerve growth factor (NGF). Then, through a remote magnetic actuation, highly aligned fibrillar gel structure embedded with anisotropically distributed coated nanoparticles, combining multiple regenerating strategies, is obtained. The effects of the multifunctional gels are examined in vitro, and in vivo in a 10-mm rat sciatic nerve injury model. The magneto-based therapeutic conduits demonstrate oriented and directed axonal growth, and improve nerve regeneration in vivo. The study of multifunctional guidance scaffolds that can be implemented efficiently and remotely provides the foundation to a novel therapeutic approach to overcome current medical obstacles for nerve injuries.     

Direct Radiation Effects on the Structure and Stability of Collagen and Other Proteins

In this review, recent progress in understanding the direct effects of radiation on the structure and stability of collagen, the most abundant protein in the human body, and other proteins is surveyed. Special emphasis is placed on the triple-helical structure of collagen, as studied by means of collagen mimetic peptides. The emerging patterns are the dose dependence of radiation processes and their abundance, the crucial role of radicals in covalent-bond formation (crosslinking) or cleavage, and the influence of the radiation energy and nature. Future research should allow fundamental questions, such as charge transfer and fragmentation dynamics triggered by ionization, to be answered, as well as developing applications such as protein-based biomaterials, notably with properties controlled by irradiation.     

Interleukin-1β Modulation of the Mechanobiology of Primary Human Pulmonary Fibroblasts: Potential Implications in Lung Repair

Pro-inflammatory cytokines like interleukin-1β (IL-1β) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1β on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1β and used Atomic Force Microscopy to unveil that IL-1β significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1β stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1β may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1β-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1β provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair.     

Functional chemical modification of a porcine acellular dermal matrix with a modified naturally derived polysaccharide crosslinker

Antibacterial and physicochemical properties are generally considered important features of the porcine acellular dermal matrix (pADM). Oxidized 2-hydroxypropyltrimethyl ammonium chloride chitosan (OHTCC) was applied to crosslink with pADM at dosages of 1, 2, 4, 8, and 16%. The properties of the crosslinked pADM (OHTCC-pADM) were evaluated. DSC and TG analysis suggested that crosslinking could promote the thermal stability, the highest T_d, and T_max of OHTCC-pADM (8%) was 80 °C and 325 °C, which has been improved by 15 °C and 13 °C, respectively. While FTIR and AFM tests indicated that the structure integrity of collagen could still be maintained. SEM tests demonstrated the sustained three-dimensional architecture of OHTCC-pADM with appropriate porosity. Moreover, OHTCC-pADM exhibited improved ability to resist collagenase degradation, the degradation rate of 4%-, 8%-OHTCC-pADM was <50%. The tensile strength of OHTCC-pADM proved to be superior compared to pADM. Furthermore, 8%-OHTCC-pADM exhibited nearly 90% antibacterial activity against Escherichia coli, and Staphylococcus aureus. In addition, the results of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide study showed that the cytocompatibility of OHTCC-pADM decreased with the increasing the amount of OHTCC, but all relative proliferation rates were above 80%. In conclusion, our study revealed that OHTCC stabilized and functionalized pADM while preserving good cytocompatibility. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47633.     

Hydrolysed collagen from Lates calcarifer skin: its acute toxicity and impact on cell proliferation and collagen production of fibroblasts

Acute toxicity in Wistar rat and the impact of hydrolysed collagen (HC) from seabass skin on in vitro cell proliferation and collagen production were studied using L929 fibroblasts. HC was rich in glycine (326 residue/1000 residue) and imino acids (196 residue/1000 residue). MALDI mass spectrum of HC showed several low MW peptides with MW range of 1050–1330 Da as the major components. Based on acute oral cytotoxicity test in Wistar rat, HC was considered as safe with LD₅₀ value higher than 5000 mg kg^?1 body. HC could promote L929 cell growth, especially when used in combination with vitamin C (VitC) at a ratio of 2:1. HC/VitC (2:1) mixture also exhibited the higher enhancement effect on collagen production of L929 cells, compared with HC or VitC alone. Thus, HC could be a promising candidate for biological applications, especially in combination with VitC, as nutraceuticals for skin care.     

聚乙烯醇/牛I型胶原支架材料的制备及评价

研究了一种聚乙烯醇(PVA)和胶原(COL)复合支架材料的制备方法。采用氨基硅烷对PVA海绵表面进行了氨基化修饰后，通过戊二醛溶液交联牛Ⅰ型胶原(COL)，最后通过赖氨酸溶液封闭，获得一种PVA/COL复合支架材料。采用扫描电镜(SEM)、X光电子能谱仪(XPS)、傅里叶红外光谱(FT-IR)等手段对支架材料的理化性能进行表征，并通过细胞实验对支架材料的生物学性能进行评价。结果表明，经过COL修饰的PVA孔隙率为21.33%，平均孔径为168.68 ?m且均匀分布，支架材料接触角为20.03°。对支架材料的生物学评价结果表明C3A细胞在复合材料上黏附良好，优于PVA组；CCK-8增殖检测结果表明细胞在复合材料上呈增殖生长趋势，与对照组PVA相比差异显著(P?0.01)。将PVA和COL复合制备得到的支架材料具有良好的理化及生物学特性，具有广阔的应用前景。