PROCEDURES

Source of Chitosan, Synthesis of Electrospinning of

Brine Shrimp
Nanosilica, Chitosan-Nanosilica- Chitosaan-Nanosilica-
(Artemia salina)
Hydroxyapatite, Hydroxyapatite Hydroxyapatite
Toxicity Assay
and SBF Reagents Composite Nanofiber

Evaluation of
Chitosan-Nanosilica- Simulated Body Fluid Disposal of Test
Hydroxyapatite (SBF) Assay Organisms
Immersed in SBF

Source of Chitosan, Nanosilica, Hydroxyapatite, and SBF Reagents

Five-hundred fifty grams of sodium bicarbonate, potassium chloride, calcium chloride

dihydrate and sodium sulfate, two-hundred fifty grams of hydroxyapatite and laboratory-grade

sodium chloride, one-hundred grams of sodium phosphate dibasic, magnesium chloride

hexahydrate and tris(hydroxymethyl)aminomethane, and fifty grams of silica (SiO2) powder with

diameters ranging from 0-2- nanometers and chitosan will be purchased from ChemLine

Scientific Enterprises, Tandang Sora, Quezon City.

Hydrochloric acid will be purchased from the local supermarket.

Synthesis of Chitosan-Nanosilica-Hydroxyapatite Composite

Chitosan will be divided into three batches that measures three grams each and will be

dissolved into 25 milliliters of dichloromethane within 24 hours. 0.25 grams of hydroxyapatite

will be weighed, put into a vial and dissolved with 8.33 milliliters of methanol (following the
ratio of DCM and methanol 3:1). Varying amounts of nanosilica in line with the literature of

Constantino (2015) and Pilic, Radusin, Ristic, Silvestre, Lazic, Baros and Duraccio (2015) will

be added to the hydroxyapatite-methanol mixture. The hydroxyapatite-methanol-nanosilica

mixture will be mixed with the chitosan solution until the mixture clears up.

Electrospinning of Chitosaan-Nanosilica-Hydroxyapatite Nanofiber

A 22-sized needle syringed will be used as a container for the polymer composite. The

syringe will then be clamped to a non-conductor stand and an alligator clip connected to a high

voltage source will be attached to the needle. The source whose voltage is 20 kilovolts will be

used. The rate of flow of the solution to the tip of the needle will be controlled by the syringe

pump. Then 15 centimeters vertically below the syringe needle, an electronically-grounded

aluminum foil will be used as the collection point of the electro spun nanofibers. The process is

being done in Technical Education and Skills Development Authority (TESDA).

Brine Shrimp (Artemia salina) Toxicity Assay

Brine Shrimp (Artemia salina) Toxicity Bioassay

Brine shrimp (Artemia salina) eggs will be acquired from a local store of

aquaculture. For the hatching of the eggs artificial seawater will be prepared by dissolving 37.00

grams of sea salt in 1 liter of distilled water. It will be put in chamber dedicated for the

hatchlings with a division for the covered area where the eggs will be placed and a lit area to

attract the hatched shrimps. The allotted time for the eggs to hatch and mature into larvae is two

days. The 10 larvae will then be placed in test tubes containing 4 milliliters of artificial sea

water. Its volume will be adjusted 5 milliliters of sea water in each test tube.
 Three replications of the same preparation will be used but in each test tube will be

artificial sea water mixed with 25 grams of chitosan-nanosilica-hydroxyapatite.

Another 3 replications are made to be used as a positive control. In each replication, 10

shrimps are placed inside the test tube containing 4 milliliters of vincristine sulfate.

The test tubes would be left under a lamp uncovered. Then, the surviving number of

hatchlings were counted and recorded after 24 hours. The experiment is being done within the

school’s premises.

Disposal of Test Organisms

The brine shrimps will be treated for 24 hours with a 10% bleach (NaClO) solution that

has a ratio of 1:9 for every culture medium. It will then be rinsed until the bleach odor has been

removed.

Simulated Body Fluid (SBF) Assay

By dissolving appropriate amounts of KCl, NaCl, NaHCO3, MgCl2.6H2O, H2O,

(CH2OH)3 CNH2, 2H2O, CaCl2, NaSO4, HCl, Na2HPO4, the SBF solution will be acquired and

each will be dissolved in 700 milliliters of distilled water. For adjusting the pH level for the

preparation of the SBF solution 40 milliliters of 1MHCl will be used. A 15-milliliter sample

of the 1MHCl solution will be added before the reagent CaCl2.H2O to prevent the slight

turbidity of the solution. The leftover HCl solution will be used in the next process called

titration. The solution’s temperature will be increased to 37°C after adding the reagent

(CH2OH)3 CNH2. It will be titrated with 1M HCl to a pH of 7.4 at 37°C, and the solution will

be diluted continuously with deionized water until the final volume equals 1 liter.
Evaluation of Chitosan-Nanosilica-Hydroxyapatite Immersed in SBF

In the preliminary assessment and visual determination of fiber uniformity a handheld

microscope will be used.

In the determination of the chemical composition and bonding sites of the nanofibers a

Fourier Transform Infrared Spectroscopy, in the range of 400-4000 cm-1 with a resolution of 4-1,

will be used.