Genetically engineered silk–collagen-like copolymer for biomedical applications: Production,characterization and evaluation of cellular response

Genetically engineered protein polymers (GEPP) are a class of multifunctional materials with precisely controlled molecular structure and property profile. Representing a promising alternative for currently used materials in biomedical applications, GEPP offer multiple benefits over natural and chemically synthesized polymers. However, producing them in sufficient quantities for preclinical research remains challenging. Here, we present results from an in vitro cellular response study of a recombinant protein polymer that is soluble at low pH but self-organizes into supramolecular fibers and physical hydrogels at neutral pH. It has a triblock structure denoted as C₂S^H₄₈C₂, which consists of hydrophilic collagen-inspired and histidine-rich silk-inspired blocks. The protein was successfully produced by the yeast Pichia pastoris in laboratory-scale bioreactors, and it was purified by selective precipitation. This efficient and inexpensive production method provided material of sufficient quantities, purity and sterility for cell culture study. Rheology and erosion studies showed that it forms hydrogels exhibiting long-term stability, self-healing behavior and tunable mechanical properties. Primary rat bone marrow cells cultured in direct contact with these hydrogels remained fully viable; however, proliferation and mineralization were relatively low compared to collagen hydrogel controls, probably because of the absence of cell-adhesive motifs. As biofunctional factors can be readily incorporated to improve material performance, our approach provides a promising route towards biomedical applications.     

Leaching of Titanium Dioxide Nanomaterials from Agricultural Soil Amended with Sewage Sludge Incineration Ash: Comparison of a Pilot Scale Simulation with Standard Laboratory Column Elution Experiments

Nanoscale titanium dioxide (nTiO₂ (Hombikat UV 100 WP)) was applied to sewage sludge that was incinerated in a large-scale waste treatment plant. The incineration ash produced was applied to soil as fertilizer at a realistic rate of 5% and investigated in pilot plant simulations regarding its leaching behavior for nTiO₂. In parallel, the applied soil material was subject to standard column leaching (DIN 19528) in order to test the suitability of the standard to predict the leaching of nanoscale contaminants from treated soil material. Relative to the reference material (similar composition but without nTiO₂ application before incineration) the test material had a total TiO₂ concentration, increased by a factor of two or 3.8 g/kg, respectively. In contrast, the TiO₂ concentration in the respective leachates of the simulation experiment differed by a factor of around 25 (maximum 91.24 mg), indicating that the added nTiO₂ might be significantly mobilisable. Nanoparticle specific analysis of the leachates (spICP-MS) confirmed this finding. In the standard column elution experiment the released amount of TiO₂ in the percolates between test and reference material differed by a factor of 4 to 6. This was also confirmed for the nTiO₂ concentrations in the percolates. Results demonstrate that the standard column leaching, developed and validated for leaching prediction of dissolved contaminants, might be also capable to indicate increased mobility of nTiO₂ in soil materials. However, experiments with further soils are needed to verify those findings.     

Effects of Graphene Oxide and Crumb Rubber on the Fresh Properties of Self-Compacting Engineered Cementitious Composite Using Response Surface Methodology

Graphene oxide-modified rubberized engineered cementitious composite (GO-RECC) is attracting the attention of researchers because of the reported benefits of the GO and crumb rubber (CR) on the strength and deformation properties of the composite. While it is well established that GO negatively affects the workability of cementitious composites, its influence on the attainment of the desired self-compacting (SC) properties of ECC has not yet been thoroughly investigated, especially when combined with crumb rubber (CR). In addition, to simplify the number of trial mixes involved in designing SC-GO-RECC, there is a need to develop and optimize the process using Design of Experiment (DOE) methods. Hence, this research aims to investigate and model using response surface methodology (RSM), the combined effects of the GO and CR on the SC properties of ECC through the determination of T₅₀₀, slump flow, V-funnel, and L-box ratio of the SC-GORECC as the responses, following the European Federation of National Associations Representing for Concrete (EFNARC) 2005 specifications. The input factors considered were the GO by wt.% of cement (0.02, 0.04, 0.06, and 0.08) and CR as a replacement of fine aggregate by volume (5, 10, and 15%). The results showed that increasing the percentages of GO and CR affected the fresh properties of the SC-GORECC adversely. However, all mixes have T₅₀₀ of 2.4 to 5.2 s, slump flow of 645 to 800 mm, V-funnel time of 7.1 to 12.3 s, and L-box ratio (H2/H1) of 0.8 to 0.98, which are all within acceptable limits specified by EFNARC 2005. The developed response prediction models were well fitted with R² values ranging from 91 to 99%. Through the optimization process, optimal values of GO and CR were found to be 0.067% and 6.8%, respectively, at a desirability value of 1.0.     

Experimental Investigation on Bending Behavior of Existing RC Beam Retrofitted with SMA-ECC Composites Materials

In order to realize the self-centering, high energy consumption, and high ductility of the existing building structure through strengthening and retrofit of structure, a method of reinforced concrete (RC) beam strengthened by using Shape Memory Alloy (SMA) and Engineered Cementitious Composites (ECC) was proposed. Four kinds of specimens were designed, including one beam strengthened with enlarging section area of steel reinforced concrete, one beam strengthened with enlarging section area of SMA reinforced concrete, beam strengthened with enlarging section area of SMA reinforced ECC, and beam strengthened with enlarging section area of steel reinforced ECC; these specimens were manufactured for the monotonic cycle loading tests study on its bending behavior. The influence on the bearing capacity, energy dissipation performance, and self-recovery capacity for each test specimens with different strengthening materials were investigated, especially the bending behavior of the beams strengthened by SMA reinforced ECC. The results show that, compared with the ordinary reinforced concrete beams, strengthening existing RC beam with enlarging section area of SMA reinforced ECC can improve the self-recovery capacity, ductility, and deformability of the specimens. Finally, a revised design formula for the bending capacity of RC beams, strengthened with enlarging sections of ECC, was proposed by considering the tensile capacity provided by ECC, and the calculated values are in good agreement with the experimental value, indicating that the revised formula can be well applied to the beam strengthening with enlarging section of SMA-ECC Materials.     

破骨细胞在骨组织工程中的意义及其研究策略

破骨细胞是骨吸收细胞,在骨塑造和重塑中发挥重要作用。目前在破骨细胞对成骨细胞调节方面的影响了解很少。最近的研究表明在骨发育过程中破骨细胞的缺乏会导致骨基质排列紊乱、矿化减少、成骨细胞行为异常。破骨细胞在骨组织工程的引入将会解决骨组织工程面临的许多问题。破骨细胞前体在组织工程化骨上生成破骨细胞是一条生理、实际的破骨细胞引入途径。     

Enzymatic synthesis of myricetin 3-O-galactoside through a whole-cell biocatalyst

Objective: Myricetin 3-O-galactoside is an active compound with pharmaceutical potential. The insufficient supply of this compound becomes a bottleneck in the druggability study of myricetin 3-O-galactoside. Thus, it is necessary to develop a biosynthetic process for myricetin 3-O-galactoside through metabolic engineering. Methods: Two genes OcSUS1 and OcUGE1 encoding sucrose synthase and UDP-glucose 4-epimerase were introduced into BL21(DE3) to reconstruct a UDP-D-galactose (UDP-Gal) biosynthetic pathway in Escherichia coli. The resultant chassis strain was able to produce UDP-Gal. Subsequently, a flavonol 3-O-galactosyltransferase DkFGT gene was transformed into the chassis strain producing UDP-Gal. An artificial pathway for myricetin 3-O-galactoside biosynthesis was thus constructed in E. coli. Results: The obtained engineered strain was demonstrated to be capable of producing myricetin 3-O-galactoside, reaching 29.7 mg/L. Conclusion: Biosynthesis of myricetin 3-O-galactoside through engineered E. coli could be achieved. This result lays the foundation for the large-scale preparation of myricetin 3-O-galactoside.     

激光近形制造钛合金试件力学性能的初步研究

目的 比较激光近形制造技术和铸造技术制作的钛合金试件的材料性能,探索激光快速成形此新工艺在口腔修复领域中的应用.方法 以-200～325目气雾化法制备的球形钛合金粉末为原料,应用激光近形制造技术制作钛合金试件,测试其在拉伸试验中的抗拉强度、屈服强度,三点弯曲试验中的弯曲弹性模量、抗弯强度,以及维氏显微硬度,并与铸造技术制作的钛合金试件进行对比;观察两种不同加工方法制作的钛合金试件的组织结构,以及拉伸断口的微观形貌特征.结果 激光近形制造技术制作钛合金试件,成形速度快,成形件无缺陷,其抗拉强度、屈服强度、弯曲弹性模量、抗弯强度、维氏显微硬度分别为(1 056.92±61.69)N/mm2、(62.65±61.91)N/mm2、(133 722.62±3 618.18)MPa、(2 313.42±129.16)MPa、376.24±14.88.激光近形制造试件的抗拉强度、屈服强度显著高于铸造钛合金试件.金相显微镜显示激光近形制造钛合金试件的组织为针状细枝晶的微晶结构,拉伸断口呈现均匀细致的韧窝形貌,发生韧性断裂.结论 激光近形制造技术可以满足口腔修复的加工要求,但需要进一步研究其成形性能及提高精度.     

利用软骨细胞膜片技术在山羊皮下构建软骨样组织的研究

目的 探讨软骨细胞膜片技术在大型哺乳动物体内构建软骨的优越性.方法 以山羊耳廓软骨细胞为种子细胞,分别利用细胞膜片技术(实验组)和细胞复合PGA/PLA支架材料的方法(对照组)构建软骨组织.体外培养4周回植到动物腹部皮下,分别于回植前、回植后4周及回植后8周时采集标本,观察比较两组软骨形成情况.结果 体外培养4周后,两组均形成一定量的软骨样组织,但对照组可见未降解的材料纤维;体内构建4周后,实验组形成成熟软骨样组织,对照组为纤维结缔组织替代,伴有大量炎性细胞浸润;体内培养8周时,实验组较4周前无明显变化,对照组体积明显缩小,仍为纤维结缔组织.结论 软骨细胞膜片技术与细胞复合PGA/PLA支架材料构建组织工程软骨方法相比,原代细胞用量少,体外培养时间短,在大动物体内能形成成熟软骨,具有更广泛的临床应用前景.     

比较带蒂筋膜瓣包裹接种自体红骨髓的组织工程骨修复骨缺损时促血管化成骨与膜诱导成骨作用

目的比较带蒂筋膜瓣包裹组织工程骨在修复骨缺损时促血管化成骨与膜诱导成骨的作用,为临床修复骨缺损提供参考依据。方法采用兔自体红骨髓(autologous red bone marrow,ARBM)及含重组人BMP-2的骨诱导活性材料构建组织工程骨。将4～5月龄新西兰大白兔60只(体重2.0～2.5 kg),随机分为A、B、C 3组,A组16只,B、C组各22只。制备长1.5 cm的右尺骨长段骨-骨膜完全缺损模型,A组于骨缺损处植入组织工程骨;B、C组在骨缺损邻近处制备一筋膜瓣包裹组织工程骨后植入骨缺损,B组将筋膜瓣蒂部切断形成游离筋膜瓣,C组为带蒂筋膜瓣。术后4、8、12、16周,每组随机取4只实验动物,取骨缺损区组织进行大体观察、组织学及免疫组织化学染色观察;8、12、16周B、C组各另取2只实验动物行腋动脉墨汁灌注观察。结果大体观察示,术后4、8周A组有纤维结缔组织形成,B、C组筋膜瓣形成类似骨膜样纤维组织,其中B组有软骨样组织形成,C组有新生骨形成;12、16周A组骨痂形成少,B组新生骨较多,C组骨干形成。组织学观察示,术后4、8周A、B组新生血管和骨小梁极少,C组血管丰富且成熟骨小梁及软骨组织形成;12、16周A组新生血管及骨小梁仍较少,B组血管数量较多且成熟骨小梁显著增加,髓腔结构形成但闭阻;C组血管数量减少,成熟骨结构形成,骨髓腔再通。免疫组织化学染色示,C组各时间点CD105、CD34、Ⅷ因子表达均高于A、B组。骨形态计量分析显示,术后各时间点C组新生骨小梁体积明显大于A、B组(P<0.05);A、B组间除4周差异无统计学意义(P>0.05)外,其余时间点差异均有统计学意义(P<0.05)。血管图像分析显示术后各时间点C组骨修复区内血管再生面积比值明显大于A、B组(P<0.05)。墨汁灌注检查示,各时间点B组成骨区为稀疏的墨染区;C组成骨区墨染数量多且较密集,8周达高峰,之后逐渐减少。结论 带蒂筋膜瓣包裹复合自体ARBM的组织工程骨,早期以促血管化成骨作用占主导地位,后期血管化成骨作用逐渐消失,以膜诱导成骨作用为主。