The polysaccharide was precipitated by mixing the ESM-EH supernatant with three volumes of cold absolute alcohol (≥ 99.5%, Sigma–Aldrich, Inc.) and then incubated at 4 °C overnight. After centrifugation at 2,850 xg, 4 °C for 20 min, the pellet was harvested and dissolved with one volume of NaCl (1.5 M). Three additional volumes of ethanol were added, and the solution was centrifuged again26 (link),27 (link). The crude extract of polysaccharide pellet was resolved with DD water and then freeze-dried, providing a fraction named ESM-C. For further purification26 (link),27 (link), ESM-C (20 mg) was dissolved in 1 mL DD water and purified by size exclusion chromatography using an HW-65 column (TSK-gel, 10 kDa-1000 kDa, 1.6 diameters × 90 cm height) to make three polysaccharide fractions, named ESM-C-A (11.3 mg), ESM-C-B (0.1 mg), and ESM-C–C (0.1 mg) (Fig. S4). The isolated carbohydrate fractions were detected by the phenol–sulfuric acid method (see Supplemental information ).
Absolute alcohol
Manufactured by Merck Group
Sourced in United States, Germany
Absolute alcohol is a highly purified form of ethanol. It contains minimal amounts of water and is commonly used as a solvent or reagent in laboratory settings. The product's core function is to provide a high-purity alcohol solution for various analytical and experimental applications.
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Lab products found in correlation
Soy lecithin, by Yuanye Bio-Technology (2 mentions)
Xylene, by Merck Group (2 mentions)
Acetic acid ch3cooh, by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Anhydrous tetrahydrofuran, by Merck Group (1 mentions)
Ammonium hydroxide solution, by Merck Group (1 mentions)
Irgacure 819, by Merck Group (1 mentions)
2 isocyanatoethyl methacrylate, by Merck Group (1 mentions)
Iron 2 chloride tetrahydrate, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Tetraethyl orthosilicate, by Merck Group (1 mentions)
Iron 3 chloride hexahydrate, by Merck Group (1 mentions)
Titanium 4 isopropoxide, by Merck Group (1 mentions)
Dibutyltin dilaurate, by Merck Group (1 mentions)
Saturated picric acid, by Merck Group (1 mentions)
Sirius red f3ba, by Merck Group (1 mentions)
Microtome, by Leica (1 mentions)
H e staining kit, by Abcam (1 mentions)
24 protocols using absolute alcohol
1
Polysaccharide Purification and Fractionation
2
Extraction and Analysis of WBM
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WBM was prepared according to our previous study [14 (link)]. In brief, the extract was prepared by drying the WBM at low temperatures (20–25°C). Powdered samples of WBM were ground and stored at −20°C. Water and WBM powder were mixed at room temperature for 24 hours. To get rid of any residues, the suspension was centrifuged at 13,000 g at 4°C for 10 minutes. After freeze-drying, the supernatant was treated with ethanol at room temperature for 24 hours while it was shaken. Under vacuum, the supernatant from the centrifuged samples was concentrated and then stored at −20°C. Extracts were dissolved in absolute alcohol (Sigma-Aldrich, St. Louis, MO, USA) before analysis.
3
Electrospinning of P34HB Scaffolds with Lecithin
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In this study, 0.8 g macromolecule compound P34HB (Bluepha, China) was dissolved in 10 mL HFIP (Merck, Germany) to obtain 8% (w/v) mixed polymer solution for electrospinning. After stirred overnight with a magnetic stirrer, the polymer solution was loaded into a standard syringe attached with a 21 G blunted stainless-steel needle. A high voltage of 15 kV was used and the mixed polymer solution was pumped to form a Taylor pendant drop which was then elongated as a fiber under electric field. A rotating collector (SS-2535, Ucalery) with a speed of 120 rpm was applied and the distance between the roller and needle tip was adjusted at 15 cm to collect the aligned fibers. Soy lecithin (Yuanye, China) solution at a concentration of 10 μg mL−1 (ref. 40 (link)) was prepared by dissolved in absolute alcohol (Sigma-Aldrich, China). The electrospun scaffolds collected on the rotating collector were soaked in Soy lecithin solution for 10 min. Finally, electrospun P34HB fiber scaffolds as well as the scaffolds with lecithin were dried overnight in vacuum to evaporate the residual organic solvent. Hence, in this paper, the pure P34HB scaffolds and electrospun P34HB fiber scaffolds with the lecithin are denoted as P34HB and P34HB/lec.
4
Extraction and Preparation of WBM Compounds
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WBM dried at low temperature was used to prepare the extract. The WBM samples were ground into a powder and stored at -20°C. WBM powder was incubated with water at room temperature for 24 h with shaking. The suspension was centrifuged at 13,000 g and 4°C for 10 min to remove any residues. The supernatant was freeze-dried, and the lyophilized powder was incubated with ethanol at room temperature for 24 h with shaking. The samples were centrifuged at 13,000 g and 4°C for 10 min, and the supernatant was concentrated under vacuum and stored at -20°C. The concentrated extract was dissolved in Absolute alcohol (≧99.8%) before analysis. Absolute alcohol was purchased from Sigma-Aldrich (St. Louis, MO, USA). α-ESA purchased from Cayman Chemical (Ann Arbor, MI, USA) was dissolved in Absolute alcohol. LPS (obtained from Escherichia coli serotype 055:B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA; L-2880).
5
Synthesis of Magnetic Polymer Nanocomposites
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Titanium(IV) isopropoxide (TTIP), glacial acetic acid, tetraethyl orthosilicate (TEOS), iron(II) chloride tetrahydrate, iron(III) chloride hexahydrate, absolute alcohol, ammonium hydroxide solution, poly(tetrahydrofuran) (PTHF, Mn ≈ 2000 g/mol), 2-isocyanatoethyl methacrylate, tetrahydrofuran anhydrous (THF), dibutyltin dilaurate and Irgacure 819 were purchased from Sigma Aldrich Chemical Co. and used without further purification.
6
Picrosirius Staining for Collagen Visualization
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Hamster cheek pouch tissue sections (2 mm thick) were subjected to the picrosirius staining protocol, as previously described in Reference [10 (link)]. Briefly, sections were deparaffinized and rehydrated, immersed in saturated picric acid (Finoric LLC, Houston, TX, USA) (2 g/100 mL) for 10 min. Sections were then stained for 30 min in a 0.1 per cent solution of Sirius red (0.1 g of Sirius red F3BA (Sigma-Aldrich, St. Louis, MO, USA), in 100 mL of saturated picric acid). This was followed by rinsing with tap water, and re-immersion in picric acid for 10 min. Slides were then dehydrated in absolute alcohol (Sigma-Aldrich®, Saint Louis, MO, USA), clarified in xylene (Sigma-Aldrich®, Saint Louis, MO, USA), and mounted in synthetic resin (Showa Denko k.k.®, Tokyo, Japan). Using polarized microscopy (Olympus, New York, NY, USA), this technique resulted in a red or yellow birefringent appearance of type I collagen.
7
Hematoxylin and Eosin Staining of Tissue Sections
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Hematoxylin and eosin (H & E) staining was performed to identify the nuclei and cytoplasm of the tissue [39 (link)]. To stain the tissues, the cultured tissues were prepared into paraffin blocks. The prepared paraffin blocks were cut into 10 μm-thick sections using a Microtome (Leica Biosystems, RM2165, Nussloch, Germany), and the sections were deparaffinized and placed on slides. The tissues were treated with Hematoxylin (Modified Mayer’s Solution) and Eosin Y solution from the H & E Staining Kit (Abcam, Cat. No. ab245880, Cambridge, UK). The tissue was washed with absolute alcohol (Sigma-Aldrich, Cat. No. 64-17-5, St. Louis, MO, USA), and the stained tissue was analyzed under a confocal microscope.
8
Quantifying Airway Collagen Deposition
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Whole lungs were excised and inflated to 10 cm H2O pressure with 10% formalin (Sigma, St. Louis, MO) to preserve pulmonary architecture. Lungs were embedded in paraffin, and sections were cut (5 μm) and processed for hematoxylin and eosin staining. Rehydrated lung sections were stained with Picro-Sirius Red Solution (Abcam, Cambridge, United Kingdom) to determine bronchial airway collagen deposition according to the manufacturer’s instructions. Briefly, the rehydrated lung sections were incubated with Picro-Sirius Red Solution for 60 min at RT, then rinsed twice by 0.5% Acetic Acid Solution (Thermo Fisher Scientific, Rockford, IL) and once by absolute alcohol (Sigma, St. Louis, MO). The slides were cleared and dehydrated in absolute alcohol, mounted with mounting medium (Thermo Fisher Scientific, Rockford, IL) and cover slips. The images were taken by EVOS XL imaging system (Life technologies, United States). For quantitative histology, airways were grouped by size in diameter of 100–200 μm, the percentage of collagen in each image were measured using ImageJ (NIH, United States).
9
Raphanus sativus L. Seed Extraction
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Sodium alginate was purchased from DAEJUNG Korea. The seeds of a Raphanus sativus L. plant were purchased from a local store. Absolute alcohol was obtained from Sigma Aldrich with a purity level of 99.9%. Sodium hydroxide (NaOH) and acetic acid (CH3COOH) and NaCl were obtained from Sigma Aldrich (St. Louis, MI, USA). Water from a Millipore Milli®-Q system (Merck KGaA, Darmstadt, Germany) was utilized for the synthesis of the gels and solutions.
10
Characterizing Scaffold Porosity and Degradation
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The porosity of the scaffolds (M) was measured by the soaking method. We calculated the volume (V) and weighed the mass (M0) of the scaffold. The sample was immersed in absolute alcohol (Sigma-Aldrich, USA) for 24 h, and the pores of the scaffold were filled under pressure. The samples were removed and weighed (M1). The porosity of each scaffold was calculated as follows: M (%) = (M1 − M0)/V × 100% (M represents the porosity of each scaffold; M0 represents the initial weight of the scaffold; M1 represents the weight of scaffolds after being immersed; V represents the volume of the scaffold).
The general evaluation of the degradation rates of the scaffolds was performed in PBS and was named the weight loss method [25 (link)]. After being dried and weighed (W0), PLA and PLA/HA scaffolds (n = 4 in each group) were placed in PBS, replaced every week, and heated in a water bath at 37 °C. At each time point (1–8 weeks), samples were collected, dried, and weighed (W1). The degradation rate of the material was calculated as follows: WT (%) = (W0 − W1)/W0 × 100% (WT represents the degradation rate of the scaffold; W0 represents the initial weight of the scaffold; W1 represents the weight of the scaffold after drying).
The general evaluation of the degradation rates of the scaffolds was performed in PBS and was named the weight loss method [25 (link)]. After being dried and weighed (W0), PLA and PLA/HA scaffolds (n = 4 in each group) were placed in PBS, replaced every week, and heated in a water bath at 37 °C. At each time point (1–8 weeks), samples were collected, dried, and weighed (W1). The degradation rate of the material was calculated as follows: WT (%) = (W0 − W1)/W0 × 100% (WT represents the degradation rate of the scaffold; W0 represents the initial weight of the scaffold; W1 represents the weight of the scaffold after drying).
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