scholarly journals 4418 Optimization and Validation of a Silk Scaffold-Based Neural Tissue Construct

2020 ◽  
Vol 4 (s1) ◽  
pp. 13-14
Author(s):  
Ben Jiahe Gu ◽  
Dennis Jgamadze ◽  
Guoming (Tony) Man ◽  
Han-Chiao Isaac Chen
Keyword(s):  
Silk Fibroin ◽  
Cortical Neurons ◽  
Primary Motor Cortex ◽  
Neural Tissue ◽  
Sprague Dawley ◽  
Rock Inhibitor ◽  
Cortical Injury ◽  
Time Points ◽  
Tissue Construct

OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

Double coating of graphene oxide–polypyrrole on silk fibroin scaffolds for neural tissue engineering

2020 ◽  
Vol 35 (3) ◽  
pp. 216-227
Author(s):  
Yuqing Wang ◽  
Haoran Yu ◽  
Haifeng Liu ◽  
Yubo Fan
Keyword(s):  
Electrical Conductivity ◽  
Tissue Engineering ◽  
Graphene Oxide ◽  
Silk Fibroin ◽  
Electrochemical Property ◽  
Neural Tissue ◽  
Neural Regeneration ◽  
Neural Tissue Engineering ◽  
Double Coating

The desired scaffolds for neural tissue engineering need to have electrical conductivity. In this study, we doubly coated graphene oxide and polypyrrole on silk fibroin scaffolds (SF@GO-PPY) by a facile method to improve its electrical conductivity. The graphene oxide–polypyrrole double coating was distributed homogeneously on silk fibroin scaffolds. Compared with silk fibroin scaffolds, the SF@GO-PPY scaffold showed higher electrical conductivity, electrochemical property, mechanical property, and thermal stability. The π–π stacking interaction between polypyrrole and graphene oxide might contribute to the superior conductive and electrochemical property of the SF@GO-PPY scaffold. Moreover, in vitro cell experiment carried out on SH-SY5Y cells showed no cytotoxicity of all the scaffolds. Thus, the results indicated that the SF@GO-PPY scaffold might be a suitable candidate for the application in neural regeneration field.

scholarly journals Cerebroprotective effect of xantohumol sensu experimental stroke model: study design and preliminary results

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Aigul Saitgareyeva ◽  
Leyla Akhmadeyeva
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Glucose Deprivation ◽  
Experimental Stroke ◽  
Control Group ◽  
Stroke Model ◽  
Sprague Dawley ◽  
Preliminary Results

The objective of our study was to evaluate the cerebroprotective effect of xanthohumol (ХN) on experimental models of acute ischemic stroke in vivo and in vitro. Materials and methods. We used middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) as in vivo and in vitro models. Our study subjects were Sprague-Dawley rats, which were randomly assigned to three groups: the control group and two MCAO groups with and without XN. The primary culture of cortical neurons was obtained from newborn rats. We employed the Bederson test and the corner test to evaluate neurological disorders. Results. The preliminary results indicated a possible cerebroprotective effect of XN in an ischemic stroke model. Conclusion. Preventive administration of XN before cerebral ischemia in an experiment can effectively reduce the volume of cerebral infarction and improve neurologic deficit 24 hours after MCAO.

scholarly journals Silk Fibroin Microneedle Patches for the Treatment of Insomnia

Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2198
Author(s):  
Zhenzhen Qi ◽  
Jiaxin Cao ◽  
Xiaosheng Tao ◽  
Xinyi Wu ◽  
Subhas C. Kundu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Silk Fibroin ◽  
Drug Concentration ◽  
Blood Concentration ◽  
The Body ◽  
Peak Time ◽  
Sprague Dawley ◽  
Transformation Mechanism

As a patient-friendly technology, drug-loaded microneedles can deliver drugs through the skin into the body. This system has broad application prospects and is receiving wide attention. Based on the knowledge acquired in this work, we successfully developed a melatonin-loaded microneedle prepared from proline/melatonin/silk fibroin. The engineered microneedles’ morphological, physical, and chemical properties were characterized to investigate their structural transformation mechanism and transdermal drug-delivery capabilities. The results indicated that the crystal structure of silk fibroin in drug-loaded microneedles was mainly Silk I crystal structure, with a low dissolution rate and suitable swelling property. Melatonin-loaded microneedles showed high mechanical properties, and the breaking strength of a single needle was 1.2 N, which could easily be penetrated the skin. The drug release results in vitro revealed that the effective drug concentration was obtained quickly during the early delivery. The successful drug concentration was maintained through continuous release at the later stage. For in vivo experimentation, the Sprague Dawley (SD) rat model of insomnia was constructed. The outcome exhibited that the melatonin-loaded microneedle released the drug into the body through the skin and maintained a high blood concentration (over 5 ng/mL) for 4–6 h. The maximum blood concentration was above 10 ng/mL, and the peak time was 0.31 h. This system indicates that it achieved the purpose of mimicking physiological release and treating insomnia.

scholarly journals Evaluating the Safety and Potential Risks of Food Allergy of Silk Fibroin Derived from Bombyx mori Cocoons

2020 ◽  
Author(s):  
Sezin Yigit ◽  
Nadia Hallaj ◽  
James Sugarman ◽  
Lester Chong ◽  
Samantha Roman ◽  
...  
Keyword(s):  
Silk Fibroin ◽  
Sprague Dawley ◽  
Bacterial Strains ◽  
Sprague Dawley Rats ◽  
Allergenic Potential ◽  
Oral Consumption ◽  
Protein Allergenicity ◽  
Potential Risks

Recent studies have demonstrated silk fibroin’s ability to extend the shelf life of foods by mitigating the hallmarks of spoilage, namely oxidation and dehydration. Due to the potential for this protein to become more widespread, its safety was evaluated comprehensively. First, a bacterial reverse mutation test (Ames test) was conducted in five bacterial strains. Second, an in vivo erythrocyte test was conducted with Sprague Dawley rats at doses up to 1,000mg/kg-bw/day. Third, a range-finder study was conducted with Sprague Dawley rats at the highest consumption amount given solubility and oral gavage volume constrains (500mg/kg-bw/day). Fourth, a 28-day study in Sprague Dawley rats was conducted at the 500mg/kg-bw/day amount. Fifth, an in vitro pepsin digestion assay was performed to assess the potential for protein allergenicity. Sixth, allergenic potential was further assessed using liquid chromatography-mass spectroscopy for detection of allergenic insect proteins. Seventh, the protein sequences were subjected to bioinformatic analyses. Together, these studies raise no mutagenic, carcinogenic, toxicological, or allergenic concerns with the oral consumption of silk fibroin.

scholarly journals A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Sui-Yi Xu ◽  
Yong-Min Wu ◽  
Zhong Ji ◽  
Xiao-Ya Gao ◽  
Su-Yue Pan
Keyword(s):  
Cortical Neurons ◽  
Dark Field ◽  
Complete Medium ◽  
Sprague Dawley ◽  
Modified Technique ◽  
Fetal Rat ◽  
Rat Cortical Neurons ◽  
Sequential Digestion ◽  
Medium Change

The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM) to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five daysin vitro(DIV); cell growth was still substantial at 14 DIV. Neurons were identified byβ-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting ofβ-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Evaluation of Structure-Controlled Silk Fibroin Coatings for Orthopedic Infection and In-Situ Osteogenesis: In Vitro and In Vivo

2020 ◽  
Author(s):  
Wenhao Zhou ◽  
Teng Zhang ◽  
Jianglong Yan ◽  
QiYao Li ◽  
Panpan Xiong ◽  
...  
Keyword(s):  
Silk Fibroin ◽  
Orthopedic Infection

scholarly journals Anti-Inflammatory Properties of Injectable Betamethasone-Loaded Tyramine-Modified Gellan Gum/Silk Fibroin Hydrogels

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1456
Author(s):  
Isabel Matos Oliveira ◽  
Cristiana Gonçalves ◽  
Myeong Eun Shin ◽  
Sumi Lee ◽  
Rui Luis Reis ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mechanical Properties ◽  
Silk Fibroin ◽  
Therapeutic Efficacy ◽  
Composite Structures ◽  
Gelation Time ◽  
Polymeric Materials ◽  
Gellan Gum ◽  
Traditional Use

Rheumatoid arthritis is a rheumatic disease for which a healing treatment does not presently exist. Silk fibroin has been extensively studied for use in drug delivery systems due to its uniqueness, versatility and strong clinical track record in medicine. However, in general, natural polymeric materials are not mechanically stable enough, and have high rates of biodegradation. Thus, synthetic materials such as gellan gum can be used to produce composite structures with biological signals to promote tissue-specific interactions while providing the desired mechanical properties. In this work, we aimed to produce hydrogels of tyramine-modified gellan gum with silk fibroin (Ty–GG/SF) via horseradish peroxidase (HRP), with encapsulated betamethasone, to improve the biocompatibility and mechanical properties, and further increase therapeutic efficacy to treat rheumatoid arthritis (RA). The Ty–GG/SF hydrogels presented a β-sheet secondary structure, with gelation time around 2–5 min, good resistance to enzymatic degradation, a suitable injectability profile, viscoelastic capacity with a significant solid component and a betamethasone-controlled release profile over time. In vitro studies showed that Ty–GG/SF hydrogels did not produce a deleterious effect on cellular metabolic activity, morphology or proliferation. Furthermore, Ty–GG/SF hydrogels with encapsulated betamethasone revealed greater therapeutic efficacy than the drug applied alone. Therefore, this strategy can provide an improvement in therapeutic efficacy when compared to the traditional use of drugs for the treatment of rheumatoid arthritis.

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