Antimicrobial Activity and Biocompatibility of Antibiotic-Loaded Chitosan Hydrogels as a Potential Scaffold in Regenerative Endodontic Treatment

IntroductionThe objective of this study was to determine the effectiveness of several antibiotic-loaded hydrogel scaffolds against Enterococcus faecalis, as well as their ability to stimulate proliferation and mineralization of dental pulp stem cells.MethodsFibrin (Fg) or chitosan-fibrin hydrogels (Ch) were prepared using 12.5 mg/mL fibrinogen and 0.4% (w/v) chitosan. Triple antibiotics, clindamycin-modified triple antibiotic paste, or double antibiotics were loaded in gels (1 mg/mL). Antibacterial effect against E. faecalis biofilm was determined by using colony-forming units (CFUs) and confocal laser scanning microscope (CLSM). Cell viability and morphology were determined by loading cells into different gels at 7 and 14 days using the water-soluble tetrazolium salt-1 cell viability assay and Live & Dead cell analysis. Mineralization was detected by using alkaline phosphatase and alizarin red staining activity.ResultsAntibiotic-loaded Fg gel and Ch gel alone without antibiotics resulted in a significant reduction in CFUs compared with the positive control (P < .05). When antibiotics were loaded in Ch gel, there were no CFUs detected in any groups (P < .05). CLSM images showed dense red areas with mostly dead bacteria on the dentin surface in antibiotic-loaded Ch groups, which showed significantly less live bacteria compared with the other groups (P < .05). Triple antibiotic-loaded Fg and Ch gels resulted in a dramatic decrease in the mineralized nodule formation compared with all other gel groups (P < .05). Ch hydrogels resulted in round cell morphology up to 7 days. Ch alone or with double antibiotic paste showed more cell spreading with spindle-shaped morphology at 14 days and higher alkaline phosphatase activity compared with other antibiotic-loaded Ch groups (P > .05).ConclusionsDouble antibiotic-loaded Ch gel appears to enhance the antibacterial properties while maintaining higher cell viability, cell spreading, and mineralization activity, compared with all the other scaffolds investigated.     

Current and novel injectable hydrogels to treat focal chondral lesions: Properties and applicability

Focal chondral lesions and early osteoarthritis (OA) are responsible for progressive joint pain and disability in millions of people worldwide, yet there is currently no surgical joint preservation treatment available to fully restore the long term functionality of cartilage. Limitations of current treatments for cartilage defects have prompted the field of cartilage tissue engineering, which seeks to integrate engineering and biological principles to promote the growth of new cartilage to replace damaged tissue. Toward improving cartilage repair, hydrogel design has advanced in recent years to improve their utility. Injectable hydrogels have emerged as a promising scaffold due to their wide range of properties, the ability to encapsulate cells within the material, and their ability to provide cues for cell differentiation. Some of these advances include the development of improved control over in situ gelation (e.g., light), new techniques to process hydrogels (e.g., multi‐layers), and better incorporation of biological signals (e.g., immobilization, controlled release, and tethering). This review summarises the innovative approaches to engineer injectable hydrogels toward cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:64–75, 2018.     

Assessing glucose and oxygen diffusion in hydrogels for the rational design of 3D stem cell scaffolds in regenerative medicine

Hydrogels are attractive biomaterials for replicating cellular microenvironments, but attention needs to be given to hydrogels diffusion properties. A large body of literature shows the promise of hydrogels as 3D culture models, cell expansion systems, cell delivery vehicles, and tissue constructs. Surprisingly, literature seems to have overlooked the important effects of nutrient diffusion on the viability of hydrogel‐encapsulated cells. In this paper, we present the methods and results of an investigation into glucose and oxygen diffusion into a silated‐hydroxypropylmethylcellulose (Si‐HPMC) hydrogel. Using both an implantable glucose sensor and implantable oxygen sensor, we continuously monitored core glucose concentration and oxygen concentration at the centre of hydrogels. We demonstrated that we could tune molecular transport in Si‐HPMC hydrogel by changing the polymer concentration. Specifically, the oxygen diffusion coefficient was found to significantly decrease from 3.4 × 10^?10 to 2.4 × 10^?10 m² s^?1 as the polymer concentration increased from 1% to 4% (w/v). Moreover, it was revealed during in vitro culture of cellularized hydrogels that oxygen depletion occurred before glucose depletion, suggesting oxygen diffusion is the major limiting factor for cell survival. Insight was also gained into the mechanism of action by which oxygen and glucose diffuse. Indeed, a direct correlation was found between the average polymer crosslinking node size and glucose parameters, and this correlation was not observed for oxygen. Overall, these experiments provide useful insights for the analysis of nutrient transport and gas exchange in hydrogels and for the development of future cellular microenvironments based on Si‐HPMC or similar polysaccharide hydrogels.     

Ocular inserts for controlled release of antibiotics

Ocular inserts impregnated with antibiotics (erythromycin and erythromycin estolate) which have sustained release characteristics were prepared, mainly for the purpose of trachoma therapy. In vitro experiments showed that the elution rate of a drug with low solubility in water (erythromycin estolate) is constant when the water content of the hydrogel insert is more than 30%. In the case of a drug with higher solubility (erythromycin), the elution rate depends on the water content. Some in vivo experiments using rabbit eyes were also reported.     

高效液相色谱法测定退障凝胶中大黄素、大黄酚的含量

目的:建立高效液相色谱法测定退障凝胶中大黄素、大黄酚含量的方法。方法:采用Waters高效液相色谱仪(Waters 2695-2487系统);Empower色谱工作站;色谱柱Kromasil C18(4.6 mm×250 mm,5μm);流动相为甲醇-0.1%磷酸溶液(82∶18);紫外检测波长254 nm;流速1.0 mL.min-1;进样量20μL。结果:大黄素在0.02～0.125μg范围内呈现良好的线性关系,大黄酚在0.072～0.360μg范围内呈现良好的线性关系,大黄素、大黄酚的加样回收率分别为99.1%,99.2%。结论:该法简便,准确,具有专属性,可用于退障凝胶的质量控制。     

Preparation of alginate hydrogels through solution extrusion and the release behavior of different drugs

Homogeneous alginate hydrogels were facilely fabricated through solution extrusion process. CaCO₃ and D-glucono-δ-lactone (GDL) were used as the gelation agents. The slow gelation of alginate was realized by the in-situ release of Ca²⁺ from CaCO₃ particles induced by hydrolysis of GDL to reduce pH. Slight gelation during the extrusion caused the enhanced strength of the alginate solutions, leading to the extrudability of the blends. This method enables to produce alginate hydrogels in a single step via extrusion, which is economically advantageous to conventional lab-scale preparation for mass production. Three different drugs, ibuprofen, acetaminophen, and methylthionine chloride, were used as model drugs to evaluate the drug release behavior of the alginate hydrogels. It was demonstrated that the drug release behavior was significantly adjusted by both the drug solubility and the ionic interaction between alginate and the drug molecule. It was shown that solution extrusion process is a feasible method to produce alginate-based drug delivery systems.     

In vivo osteogenic differentiation of human turbinate mesenchymal stem cells in an injectable in situ-forming hydrogel

Human turbinate mesenchymal stromal cells (hTMSCs) are an alternate source of adult stem cells for regenerative medicine. In this work, we demonstrated that hTMSCs are easily harvested from turbinate tissue using a minimal surgical procedure. hTMSCs showed positive expression of mesenchymal stem cell markers and proliferated at a high rate. The specific surface proteins of harvested hTMSCs were relatively tolerant of ex vivo manipulation in culture. hTMSCs exhibited osteogenic differentiation in vitro in the presence of osteogenic factors. To examine osteogenic differentiation of hTMSCs in vivo in an injectable hydrogel, cells were incorporated into a methoxy polyethylene glycol–polycaprolactone block copolymer (MPEG–PCL (MP)) solution simply by mixing. hTMSC-loaded MP solutions exhibited a temperature-dependent solution-to-gel phase transition. The hTMSC attached and grew well on in vitro- and in vivo-formed MP hydrogels. hTMSC-loaded MP solutions formed a hydrogel almost immediately upon injection into animals and the cells remained viable, even after 12 weeks. Injected hTMSCs in in situ-formed MP hydrogels differentiated into osteogenic cells, mainly in the presence of osteogenic factors. Differentiated osteoblasts were identified by Alizarin Red S, von Kossa, and alkaline phosphatase (ALP) staining, and osteonectin, osteopontin, and osteocalcin mRNA expression. To the best of our knowledge, this is the first study to show hTMSCs undergoing osteogenic differentiation in in vivo-formed MP hydrogels. In conclusion, hTMSCs could serve as adult stem cell sources and, when embedded in an in situ-formed hydrogel, may provide numerous benefits as a noninvasive alternative for bone tissue engineering applications.     

人工髓核材料(半晶聚乙烯醇水凝胶弹性体)的研制

研制一种可替代椎间盘髓核并恢复其功能的生物医用材料 ,探讨半晶聚乙烯醇水凝胶弹性体材料临床应用的可行性。聚乙烯醇 (PVA)水溶液在 - 2 0℃下冷冻 6～ 12 h,室温下融化 1～ 2 h,上述过程重复 1～ 3次 ,然后对试样进行真空脱水 ,制得人工髓核材料 (半晶 PVA水凝胶弹性体 )。差示扫描量热法 (DSC)和力学性能试验研究了 PVA水溶液浓度、真空脱水和 γ射线辐照对水凝胶 PVA的结晶度和力学性能的影响     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

Morphological alterations and distribution of occludin in rat testes after bilateral vasectomy

The aim of study was to investigate the fate and the morphology of the cells which constitute the spermatogenic line, and to determine the distribution of occludin in the testis in adult vasectomized Wistar rats. The rats were divided into two groups: control group (sham-operated) and vasectomized group. One, 3 and 6 months after sham and vasectomy operations, testis samples were examined. The weight of the testes was found to be reduced 3 and 6 months after vasectomy. There was vacuolization in the seminiferous tubules one month after vasectomy. The tubules showed severe atrophy 3 and 6 months after vasectomy. The occludin immunolabeling in the 3- and 6-month groups was weak and diffuse, and the density of the protein was found to be decreased. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in the number of haploid, diploid and tetraploid cells. This study demonstrated that vasectomy causes degeneration in the seminiferous tubules with alterations in occludin distribution with a decrease in the number of spermatogenic cells. Moreover, these alterations increase in a time-dependent manner.