基于增材制造和凝胶注模成型技术的多孔生物陶瓷支架制备与表征

目的探讨基于增材制造和凝胶注模成型技术的多孔β-磷酸三钙（TCP）生物陶瓷支架的制备方法及其表征。方法利用计算机辅助设计（CAD）软件设计支架内部孔隙结构,通过光固化快速成型技术制造相应的树脂模具,在模具中填充生物材料,待其固化后通过热分解去除树脂模具,然后对所形成的多孔β-TCP支架的微观孔隙结构特征、力学性能以及体外细胞相容性进行检测。结果多孔β-TCP支架孔隙结构与设计结构一致,孔隙率为45.1%±1.2%,孔的尺寸为300～500μm;力学性能测试表明,支架的平均抗压强度为5.3±0.8 MPa;成骨细胞能够在支架上黏附生长,支架具有良好的生物相容性。结论基于增材制造技术和凝胶注模成型工艺的多孔生物陶瓷支架制备方法,可实现支架复杂外形与内部微结构的精确控制和一体化制造。     

Modern drug delivery strategies applied to natural active compounds

Introduction: Colloidal drug delivery systems (CDDSs) are innovative carriers that have been studied in pharmaceutical field from many years to overcome unfavorable physical and chemical features of synthetic drugs. Recently the use of CDDS as carriers for phytochemicals has seen an exponential increase which, in some cases, has led to the rediscovery of ancient and forgotten natural molecules.

Area covered: This article focuses on the main features of CDDS, particularly micro- and nanoemulsions, vesicular carriers and micro- and nanoparticles, loaded with natural active compounds. A detailed review of the literature is presented, introducing the importance of these systems in terms of their capability to optimize the stability of phytochemicals, their absorption through biological membranes and their bioavailability.

Expert opinion: The delivery of phytochemicals is problematic due to poor solubility, poor permeability, low bioavailability, instability in biological milieu and extensive first-pass metabolism. Global research efforts investigating nanotechnology have attempted to overcome these limitations rediscovering and, in some cases, ‘discovering ex novo’ unexpected virtues and benefits associated to these compounds. The ‘nanotechnological approach’ can definitely enhance the pharmacokinetics and therapeutic index of natural active compounds and improve their performance in therapy.     

3D生物打印技术在组织工程支架构建与再生中的应用进展

具备个性化、精准化的3D生物打印技术构建优良生物相容性的组织工程支架,替换或修复人体中病变的组织和/或器官,在组织工程研究中极具广泛的应用前景。而作为3D打印技术的基材,如金属、生物陶瓷、高分子材料和细胞生物材料等,也因此得到了研究者的重视和研发。由金属和生物陶瓷制备的支架具有高强度和耐腐蚀性,已在骨科中得到广泛应用,而高分子材料由于其和细胞/组织的良好生物相容性及可塑性使其在软骨、矫形外科、心血管系统等组织/器官中被广泛研究。相信在不久的将来,以上述材料为基材结合3D生物打印技术构建的组织和器官,在临床上会得到应用和推广。本文就用于3D打印的生物材料及其打印技术在组织工程支架构建及组织再生中的应用进行综述。     

hns基因对大肠埃希菌生物膜形成能力的影响

目的 建立大肠埃希菌Red重组系统无痕敲除方法,探讨hns基因对大肠埃希菌生物膜形成能力的影响.方法 以大肠埃希菌MG1655为研究对象,将pKD46质粒转化入MG1655感受态细胞.以质粒pKD3为模板扩增氯霉素抗性基因片段,并将其转化入MG1655/pKD46,氯霉素抗性平板筛选阳性克隆.利用pCP20质粒删除氯霉素抗性基因,构建MG1655 hns基因缺失菌株.96孔板结晶紫染色法分析MG1655 hns基因缺失株生物膜形成能力的变化.苯酚-硫酸法检测细菌胞外多糖含量.结果 氯霉素抗性基因消除后的hns基因缺失株PCR产物长度为434 bp,测序鉴定正确,成功构建MG1655 △hns基因缺失株.MG1655△hns菌株生物膜形成能力和胞外多糖产生显著低于MG1655(P＜0.01).结论 利用Red重组技术成功构建出MG1655△hns菌株,hns基因对细菌生物膜的形成具有重要的调控作用.     

Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge

Cultured cells are dramatically affected by the micro-environment in which they are grown. In this study, we have investigated whether HepG2 liver cells grown in three dimensional (3-D) cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional (2-D) flat plastic surfaces. To enable 3-D growth of HepG2 cells in vitro, we cultured cells on 3-D porous polystyrene scaffolds previously developed in our laboratories. HepG2 cells grown in 3-D displayed excellent morphological characteristics and formed numerous bile canaliculi that were seldom seen in cultures grown on 2-D surfaces. The function of liver cells grown on 3-D supports was significantly enhanced compared to activity of cells grown on 2-D standard plasticware. Unlike their 2-D counterparts, 3-D cultures were less susceptible to lower concentrations of methotrexate. Cells grown in 3-D maintained their structural integrity, possessed greater viability, were less susceptible to cell death at higher levels of the cytotoxin compared to 2-D cultures, and appeared to respond to the drug in a manner more comparable to its known activity in vivo. Our results suggest that hepatotoxicity testing using 3-D cultures might be more likely to reflect true physiological responses to cytotoxic compounds than existing models that rely on 2-D culture systems. This technology has potential applications for toxicity testing and drug screening.     

Protein Interfacial Pocket Engineering via Coupled Computational Filtering and Biological Focusing Criterion

To engineer bio-macromolecular systems, protein–substrate interactions and their configurations need to be understood, harnessed, and utilized. Due to the inherent large numbers of combinatorial configurations and conformational complexity, methods that rely on heuristics or stochastics, such as practical computational filtering (CF) or biological focusing (BF) criterions, when used alone rarely yield insights into these complexes or successes in (re)designing them. Here we use a coupled CF–BF criterion upon an amenable interfacial pocket (IP) of a protein scaffold complexed with its substrate to undergo residue replacement and R-group refinement (R⁴) to filter out energetically unfavorable residues and R-group conformations, and focus in on those that are evolutionarily favorable. We show that this coupled filtering and focusing can efficiently provide a putative engineered IP candidate and validate it computationally and empirically. The CF–BF criterion may permit holistic understanding of the nuances of existing protein IPs and their scaffolds and facilitate bioengineering efforts to alter substrate specificity. Such approach may contribute to accelerated elucidation of engineering principles of bio-macromolecular systems. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Dehydroascorbic Acid and pGPMA Dual Modified pH-Sensitive Polymeric Micelles for Target Treatment of Liver Cancer

In clinical therapy, the poor prognosis of hepatocellular carcinoma (HCC) is mainly attributed to the failure of chemotherapeutical agents to accumulate in tumor as well as lack of potency of tumor penetration. In this work, we developed actively tumor-targeting micelles with pH-sensitive linker as a novel nanocarrier for HCC therapy. These micelles comprised biodegradable poly(ethylene glycol)-poly(aspartate) polymers, in which paclitaxel can be covalently conjugated to pAsp via an acid-labile acetal bond to form pH-responsive structures. In vitro drug release studies showed that these structures were stable in physiological condition, whereas collapsed once internalized into cells due to the mildly acidic environment in endo/lysosomes, resulting in facilitated intracellular paclitaxel release. In addition, dehydroascorbic acid and guanidinopropyl methacrylamide polymers were decorated on the surface of micelles to achieve specific tumor accumulation and tumor penetration. Cellular uptake and in vivo imaging studies proved that these micelles had remarkable targeting property toward hepatocarcinoma cells and tumor. Enhanced anti-HCC efficacy of the micelles was also confirmed both in vitro and in vivo. Therefore, this micellar system may be a potential platform of chemotherapeutics delivery for HCC therapy.     

Amphotericin B-Loaded Poly(lactic-co-glycolic acid) Nanofibers: An Alternative Therapy Scheme for Local Treatment of Vulvovaginal Candidiasis

Vulvovaginal candidiasis is an inflammation localized in the vulvovaginal area. It is mostly caused by Candida albicans. Its treatment is based on the systemic and local administration of antifungal drugs. However, this conventional therapy can fail owing to the resistance of the Candida species and noncompliance of patients. Amphotericin B-loaded poly(lactic-co-glycolic acid) nanofibers are single-use, antifungal, controlled drug delivery systems, and represent an alternative therapeutic scheme for the local treatment of vulvovaginal candidiasis. Nanofibers were characterized by analytical techniques and with an in vitro drug delivery study. In vitro and in vivo fungicidal activity of amphotericin B released from nanofibers was evaluated using the agar diffusion method and an experimental murine model of vulvovaginal candidiasis, respectively. Analytical techniques showed that amphotericin B was physically mixed in the polymeric nanofibers. Nanofibers controlled the delivery of therapeutic doses of amphotericin B for 8 consecutive days, providing effective in vitro antifungal activity and eliminated the in vivo vaginal fungal burden after 3 days of treatment and with only one local application. Amphotericin B-loaded poly(lactic-co-glycolic acid) nanofibers could be potentially applied as an alternative strategy for the local treatment of vulvovaginal candidiasis without inducing fungal resistance, yet ensuring patient compliance.     

Impact of Superdisintegrants and Film Thickness on Disintegration Time of Strip Films Loaded With Poorly Water-Soluble Drug Microparticles

Although strip films are a promising platform for delivery of poorly water-soluble drug particles via slurry casting, the effect of critical material attributes, for example, superdisintegrants (SDIs) on critical quality attributes, including film disintegration time (DT), remains underexplored. A 2-level factorial design is considered to examine the impact of the SDI type (sodium starch glycolate and croscarmellose sodium), their amount, and film thickness. SDIs were used with hydroxypropyl methylcellulose (E15LV) and glycerin solutions along with viscosity matching. Fenofibrate, a model poorly water-soluble drug, was micronized and surface modified via fluid energy milling. Significant decreases in film DT, measured using 3 different methods, were observed due to the addition of SDIs. Percentage reduction in DT was a strong function of SDI amount, and thinner films disintegrated faster. Films with either higher SDI concentrations (>9%) or films under 80 μm, exhibited fast DT (<180 s, European Pharmacopeia). All thin films (50-60 μm) exhibited immediate release (>80% in 10 min). All films achieved good content uniformity, except for those with the lowest amount of SDI, attributed to insufficient viscosity and thickness nonuniformity due to the SDI. Finally, all films achieved adequate mechanical properties, notwithstanding minor negative impact of SDIs.     

Fabrication of hybrid scaffold based on hydroxyapatite-biodegradable nanofibers incorporated with liposomal formulation of BMP-2 peptide for bone tissue engineering

In the present study, we fabricated an efficient, simple biomimetic scaffold to stimulate osteogenic differentiation of mesenchymal stem cells (MSCs). Electrospun poly L-lactic acid nanofibers were employed to mimic the nanofibrillar structure of bone proteins and coated with hydroxyapatite nanoparticles to simulate bone minerals. Thereafter, we regulated the release pattern of BMP-2 peptide through covalent attachment of an optimized liposomal formulation to the scaffold. The fabricated platform provided a sustained release profile of BMP-2 peptide up to 21?days while supporting cellular attachment and proliferation without cytotoxicity. In-vitro results confirmed the superiority of the scaffold containing liposomes through enhancement of growth and differentiation of MSCs. Ectopic bone formation model exhibited significant localized initiation of bone formation of liposome incorporated scaffold. Consequently, these findings demonstrated that our designed platform with modified release properties of BMP-2 peptide considerably promoted osteogenic differentiation of MSCs making it a unique candidate for bone regeneration therapeutics.