ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 1

Development, Characterization and Toxicity of Electrospun Polylactic Acid (PLA)-Nanosilica

(SiO2)-Cellulose Acetate Nanofiber in Deposition of Hydroxyapatite (HA) at Synthetic Body

Fluid (SBF): Potential Bioartificial Bone Tissue Scaffold

Piolo Gabriel N. Gervacio

Investigator

RESEARCH PLAN
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 2

RATIONALE

The skeletal system aids the movement, shape and stands as a foundation of the human body. It is

not static and is known of its regenerative capability, yet it still cannot hinder the problems that arise

regarding the structure of bones such as the fragility, infection and bone loss (Medline Plus, 2014).

Bone grafting techniques with altering probabilities of safety have been developed, yet

these are considered impracticable as it comes to massive bone loss and poses the threat of rejection

by the immune system and microbiological contamination (Bao, Teo, Chong, Liu, Choolani and

Chan, 2013). Although demineralized bone matrix resulted into success in animal studies, it is not

proven to effectively merge with human spine and is only suggested as a bone graft extender not

as a substitute. (Wang and Kalfas, 2015)

In response to the given complication, this study will be conducted as a resolution to the

drawbacks of the former methods. It intends to produce an innovative scaffold, tested through in

vitro procedures, as a replacement for traditional methods, discarding the need for donors and

overcoming difficulties tackled by the field of osteology.

Through this study, a path will be paved for the discovery of the abilities of polylactic acid,

nanosilica and cellulose acetate and how it can be used to benefit the field of osteology. It will

create and extend the different scientific fields continuously and initiate inquiries that will generate

promising future researches.

PROBLEMS

This study aims to determine the capabilities of Electrospun Polylactic Acid (PLA)-

Nanosilica (SiO2)-Cellulose Acetate Nanofiber as a bioartificial bone scaffold in bone tissue

 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 3

engineering via deposition of hydroxyapatite through in-vitro Synthetic Body Fluid (SBF)

immersion.

Specifically, this study is concerned with seeking answers to the following questions:

1. How can the unimmersed electrospun PLA-SiO2-CA nanofibers be described in terms of

the following:

a) Visual Determination of Fiber Uniformity,

b) Toxicity,

c) Mean Fiber Diameter; and

d) Chemical Structure?

2. How can the immersed electrospun PLA-SiO2-CA nanofibers be described in terms of the

following:

a) Mean Fiber Diameter; and

b) Hydroxyapatite Ca/P Ratio?

3. Will there be a significant difference between the unimmersed electrospun PLA-SiO2-CA

nanofibers in terms of the mean fiber diameter?

4. Will there be a significant difference between the immersed electrospun PLA-SiO2-CA

nanofibers in terms of the following:

a) Mean Fiber Diameter; and

b) Hydroxyapatite Ca/P Ratio?

5. Will there be a significant difference between the immersed and unimmersed electrospun

PLA-SiO2-CA nanofibers in terms of the mean fiber diameter?

ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 4

HYPOTHESES

H0 : There is no significant difference between the nanofiber diameter size range in the

unimmersed nanofiber treatments with different amounts of nanosilica and constant

amounts of polylactic acid and cellulose acetate

There is no significant difference between the nanofiber diameter size range in the

immersed nanofiber treatments with different amounts of nanosilica and constant amounts

of polylactic acid and cellulose acetate

There is no significant difference between the Ca/P ratio of hydroxyapatites

attained through SBF immersion of nanofiber treatments with different amounts of

nanosilica and constant amounts of polylactic acid and cellulose acetate in 96 hours.

There is no significant difference between the nanofiber diameter size range in the

unimmersed and immersed nanofiber treatments with different amounts of nanosilica and

constant amounts of polylactic acid and cellulose acetate

Ha : There is a significant difference between the nanofiber diameter size range in the

unimmersed nanofiber treatments with different amounts of nanosilica and constant

amounts of polylactic acid and cellulose acetate

There is a significant difference between the nanofiber diameter size range in the

immersed nanofiber treatments with different amounts of nanosilica and constant amounts

of polylactic acid and cellulose acetate

ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 5

There is a significant difference between the Ca/P ratio of hydroxyapatites attained

through SBF immersion of nanofiber treatments with different amounts of nanosilica and

constant amounts of polylactic acid and cellulose acetate in 96 hours.

There is a significant difference between the nanofiber diameter size range in the

unimmersed and immersed nanofiber treatments with different amounts of nanosilica and

constant amounts of polylactic acid and cellulose acetate

ENGINEERING GOALS

The study will aim to achieve the following:

 Develop an innovative biomimetic bone tissue scaffold as a replacement for risky

bone grafting and techniques.

 Determine the prominent significance of nanotechnology in the field of bone tissue

engineering.

 Solve the problem regarding osteal diseases such as fragility, infection and bone

loss.

 Benefit the researches whom this study may provide significant data for the

searching of the potential of polylactic acid (PLA), nanosilica (SiO2) and cellulose

acetate in the field of osteology.

 Give way to solutions that may eradicate the hindrances and difficulties regarding

the bone structures in the near future.

 Utilize polylactic acid, nanosilica and cellulose acetate as a medium and a

component in bone tissue engineering products.

 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 6

EXPECTED OUTCOMES

 The polymers and nanosilica would mix and the nanofiber would show similar

characteristics to those of the raw materials.

 The varied amount of nanosilica in the nanofibers would have an effect on the

mechanical properties and hydroxyapatite formation capability of each treatment.

 The varied amount of nanosilica in the nanofibers would have an effect on the

Ca/P ratio of the hydroxyapatite aggregates, which can help it reach the needed

range for a passable scaffold.

 The electrospun polylactic acid-nanosilica-cellulose acetate nanofiber scaffold

would pass as a potential bioartificial bone tissue scaffolding.

PROCEDURES

Source of Nanosilica, Cellulose Acetate, Polylactic Acid and SBF Reagents

Fifty grams of silica (SiO2) powder with diameters ranging from 0-20 nanometres and

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

hexahydrate and tris(hydroxymethyl)aminomethane, two-hundred fifty grams of cellulose acetate

and laboratory-grade sodium chloride, and five-hundred grams of sodium bicarbonate, potassium

chloride, calcium chloride dihydrate and sodium sulfate will be purchased from ChemLine

Scientific Enterprises, Tandang Sora, Quezon City.

Hydrochloric acid will be purchased from the local supermarket.

 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 7

Synthesis of Electrospun Nanosilica-Cellulose Acetate-Polylactic Acid Composite

Three batches of three grams of polylactic acid will be dissolved with 25

millilitres of dichloromethane each on a period of 24 hours (Makadia and Siegel, 2011). 0.25

grams of cellulose acetate will be weighed, put into a vial and dissolved with 8.33 millilitres 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 cellulose acetate-methanol mixture. The cellulose acetate-

methanol-nanosilica mixture will be mixed with the polylactic acid solution until it the mixture

clears up.

Electrospinning of Nanosilica-Cellulose Acetate-Polylactic Acid Nanofiber

The polymer composite will be placed in a syringe with a 22-sized needle. The syringe

will be clamped to an insulated stand and an alligator tip connected to the high voltage source

will be attached to the needle. The voltage will be adjusted at 20 kilovolts. The syringe pump

will be used as the controller of the flow rate of the polymer solution to the needle tip. The

electrospun nanofibers will be collected on an electrically-grounded aluminium foil 15

centimetres vertically below the syringe needle (Yeum, Park, Kim and Cheong, 2011).

Brine Shrimp (Artemia salina) Toxicity Assay

Brine Shrimp (Artemia salina) Toxicity Bioassay

Brine shrimp (Artemia salina) eggs will be obtained from a local fishery and

aquaculture store. Artificial seawater will be prepared by dissolving 38.00 grams of sea

salt in 1 litre of distilled water for the hatching of the shrimp eggs. The prepared
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 8

seawater will be put in a small plastic container (hatching chamber) with a divider for

the dark (covered) where the eggs will be placed and a light area to attract the hatched

shrimps. Two days will be allotted for the shrimps to hatch and mature to larvae. Ten

brine shrimp larvae will be introduced to test tubes containing 4 millilitres of artificial

seawater. The volume will be adjusted with up to 5 millilitres of artificial seawater per

test tube. The test tubes will be left uncovered under a lamp, and the surviving shrimps

will be counted and recorded after 24 hours.

Disposal of Test Organisms

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

ratio of 1:9 for every culture medium. The equipment will be rinsed until the bleach odour has

been removed (Ward’s Science, 2008).

Simulated Body Fluid (SBF) Assay

The SBF solution will be obtained by dissolving appropriate quantities of NaCl,

NaHCO3, KCl, Na2HPO4, 2H2O, MgCl2.6H2O, Na2SO4, (CH2OH)3 CNH2, CaCl2.H2O and HCl

in distilled water. The reagents will be added one by one (in the order presented in Table 2) after

the preceding has been dissolved completely in 700 millilitres of distilled water. 40 millilitres of

1M HCl solution will be used for pH adjustments in the preparation of the SBF solution. To

prevent the slight turbidity of the solution, a 15 millilitre aliquot of the 1M HCl solution will be

added before adding the sixth reagent CaCl2.H2O. The remaining HCl solution will be used in

the succeeding titration. After adding the eight reagent (CH2OH)3 CNH2, the temperature of the

solution will be raised to 37°C. It will be titrated with 1M HCl to a pH of 7.4 at 37°C, and the
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 9

solution will be diluted continuously with deionized water until the final volume equals 1 litre

(Tas, 1999).

Table 1: Chemical Composition of Synthetic Body Fluid (SBF) Solution

Order Reagent Chemical Formula Amount (grams)

1 Sodium Chloride NaCl 6.547
2 Sodium Bicarbonate NaHCO3 2.268
3 Potassium Chloride KCl 0.373
Sodium Phosphate
4 Na2HPO4.2H2O 0.178
Dibasic
Magnesium Chloride
5 MgCl2.6H2O 0.305
Hexahydrate
Calcium Chloride
6 CaCl2.2H2O 0.368
Dihydrate
7 Sodium Sulphate Na2SO4 0.710
Tris(hydroxymethyl)
8 (CH2OH)3 CNH2 6.057
aminomethane
The nanofibers (cut to lengths of 3cm x 1cm) will be exposed to 22 millilitres of SBF

solution per vial in an oven at a temperature of 37°C ± 0.4°C at a period of 96 hours, with pH

regularly checked every 24 hours (Helebrant, Jonasova and Sanda, 2001).

Evaluation of Electrospun Nanosilica-Cellulose Acetate-Polylactic Acid Nanofibers

Immersed in SBF

Handheld microscope will be used in the preliminary assessment and visual

determination of fiber uniformity.

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

of 4-1, will be used in the determination of the chemical composition and bonding sites of the

nanofibers (Liu, Yeh, Lipner, Xie, Sung, Thomopoulos and Xia, 2011).
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 10

The nanofibers immersed in SBF will be sputter-coated with gold-palladium alloy before

analysing with the scanning electron microscope to determine surface morphology and

microstructure of the nanofibers. Images of the electrospun nanosilica-cellulose acetate-

polylactic acid nanofibers will be obtained through the Scanning Electron Microscope (SEM)

operated at an accelerating voltage of 15 kilovolts (Zhang, Venugopal, El-Turki, Ramakrishna,

Su and Lim, 2008).

Energy Dispersive X-ray (EDX) Spectroscopy will be used in analysing the layer

deposited on the surface of the nanofibers, specifically calcium and phosphorus (Chavan, Bahir,

Mene, Mahabole and Khairnar, 2009).

RISKS

 Polylactic Acid

 PLA may cause mild skin and eye irritation and can react with water. Solids or

dusts from the chemical may also cause corneal injury and eye irritation due to

mechanical action. PLA ingestion can cause gastrointestinal irritation, nausea,

vomiting and diarrhoea (Velleman NV, n.d.; NatureWorks LLC, 2009). Polylactic

acid can ignite under sufficient heat or near an ignition source, however it is

stable under normal temperatures and pressures (Velleman NV, n.d.)

 Usage of personal protective equipment such as rubber gloves, safely glasses and

respirators are recommended. Waste disposal must include the use of personal

protective equipment and landfill burying with obedience to all regulations. The

chemical must be kept away from the atmospheric conditions. Avoiding dust
ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 11

accumulation needs to be done in order to avoid potential dust explosions.

(Reprefer Tech Co., 2012).

 PLA is non-hazardous and biodegradable. Adequate disposal methods include

sanitary landfill burying and incineration in accordance to the prevailing

municipal, state and federal laws and standards from local environment agencies.

Accidental release to the environment without proper measures should be avoided

(Reprefer Tech Co., 2012; Velleman NV, n.d.).

 Nanosilica

 Nanosilica is considered as an eye, skin, respiratory and gastrointestinal irritant

due to mechanical action (Sigma Aldrich, 2015). Nanosilica is non-flammable,

non-combustible and stable under normal temperatures and pressures (Nanosil,

n.d.)

 One must not swallow or inhale gas, fumes, vapour or sprays from the chemical.

Putting on of protective clothing and appropriate respiratory equipment if there is

deficient air circulation are recommended. Skin or eye contact are needed to be

avoided as much as possible (Nanosil, n.d.).

 Nanosilica is a non-hazardous waste according to EPA, RCRA 40CFR, Part 261,

1990. The material may be disposed of as a non-hazardous solid waste and

adequate methods include sanitary landfill burying in accordance to the prevailing

municipal, state and federal laws and standards from local environment agencies

(Nanosil, n.d.).
ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 12

 Cellulose Acetate

 Cellulose acetate is considered as an eye, skin, gastrointestinal and respiratory

irritant due to mechanical actions. Cellulose acetate can ignite via static charges,

but the material is stable under normal pressures and temperatures.

 One must not swallow or inhale gas, fumes, vapour or sprays from the chemical.

Putting on of protective clothing is recommended and direct contact with the

substance is needed to be avoided as much as possible. Proper bonding and

grounding procedures should be done to avoid fire hazards (ScienceLab.com,

2013).

 Cellulose acetate waste is not considered as hazardous waste under Resource

Conservation and Recovery Act (RCRA), 40 CFR 261. Waste should can be

disposed of through incineration in accordance to prevailing municipal, state and

federal laws and standards from local environment agencies (Eastman Chemical

Company, 2014).

 Methanol

 Methanol is toxic when it comes to contact with the skin, and when swallowed

and inhaled. It may also cause erythema, dermatitis, defatting in the tissues,

itching, burning, redness, tearing, blindness, drowsiness, dizziness, CNS-

depression, narcosis, coma, liver damage, headache, sleepiness, slurred speech,

blurred vision and adverse reproductive effects.

 Not breathing its vapors and come in contact with it through skin, eyes, mouth

and nose, not being near an open flame, knowing where the equipment is in case

of exposure, and wearing proper attire, with chemically resistant and safety
ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 13

equipment are the safety procedures when handling the chemical (Caltech.edu,

2006).

 Methanol should be recycled whenever possible via re-distillation or if

contaminated, controlled incineration in accordance to prevailing municipal, state

and federal laws and standards from local environment agencies (BDH, 2006).

 Dichloromethane (DCM)

 Depending on the time of exposure from the chemical, DCM can cause headache,

lightheadedness, weakness, irritability, and nausea. Exposure to higher levels of

DCM causes slurred speech, poor coordination, giddiness, confusion, lethargy,

and ataxia. Prolonged contact can cause chemical burns. Excessive inhalation may

cause a serious case of pulmonary edema. It can cause fatalities due to accidental

inhalation and skin contamination. Liver and renal dysfunctions and effects on

hematological parameters have also been reported following exposure to

methylene chloride (BDH, 2006; ToxNet, 2014).

 Usage of personal protecting tools, ensurance of appropriate air circulation and

removal of flammable/combustible materials must be done. Wearing of

impermeable clothing, gloves, safety goggles, credible and verified respirators

must be employed. Providing eye stations and shower facilities are necessary.

 Dichloromethane should be disposed of as a special waste in accordance to

prevailing municipal, state and federal laws and standards from local environment

agencies. Incineration of waste material in an EPA-approved facility is

recommended (BDH, 2006).

ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 14

 Sodium Chloride

 Sodium chloride is considered as an eye, skin, respiratory and gastrointestinal

irritant due to mechanical action. Sodium chloride is not a fire hazard and is stable

under normal temperatures and pressures.

 Splash goggles, lab coat, dust respirator, boots, gloves and a self-contained

breathing apparatus should be used while performing experiments with sodium

chloride (ScienceLab.com, 2013).

 Sodium chloride waste is not considered as hazardous waste under Resource

Conservation and Recovery Act (RCRA), 40 CFR 261. Waste should be disposed

of while observing all precautions in compliance to prevailing municipal, state

and federal laws and standards from local environment agencies

(ScienceLab.com, 2013).

 Sodium Bicarbonate

 Mild skin and eye irritation, respiratory tract irritation, thirst, abdominal pain,

gastroenteritis, and inflammation of the digestive tract may be encountered if

sodium bicarbonate is ingested, spilt in the eye and skin, or inhaled. Severe

toxicity may also affect behaviour and the central nervous system. Sodium

bicarbonate is not a fire hazard and is stable under normal temperatures and

pressures.

 Splash goggles, lab coat, dust respirator, boots, gloves and a self-contained

breathing apparatus should be used while performing experiments with sodium

bicarbonate (ScienceLab.com, 2013).

ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 15

 Sodium bicarbonate waste is not considered as hazardous waste under Resource

Conservation and Recovery Act (RCRA), 40 CFR 261. Waste should be disposed

of while observing all precautions in compliance to prevailing municipal, state

and federal laws and standards from local environment agencies

(ScienceLab.com, 2013).

 Potassium Chloride

 Potassium chloride is considered as an eye, skin, respiratory and gastrointestinal

irritant due to mechanical action. The substance, however, may be toxic to blood

and the cardiovascular system. Prolonged exposure may cause organ damage.

Potassium chloride is not a fire hazard and is stable under normal temperatures

and pressures.

 Usage of process enclosures, local exhaust ventilation, or other engineering

controls to keep airborne levels below recommended exposure limits are

recommended. Wearing of proper protective equipment must be employed

(ScienceLab.com, 2013).

 Potassium chloride waste is not considered as hazardous waste under Resource

Conservation and Recovery Act (RCRA), 40 CFR 261. Waste should be disposed

of while observing all precautions in compliance to prevailing municipal, state

and federal laws and standards from local environment agencies

(ScienceLab.com, 2013).

 Sodium Phosphate Dibasic

 Skin and eye irritation, nose and throat irritation causing coughing and wheezing

are the risks that may be encountered if sodium phosphate dibasic makes contact
ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 16

with skin and eyes and if inhaled. High and prolonged exposure to the substance

may cause a skin rash. Sodium phosphate dibasic is not a fire hazard and is stable

under normal temperatures and pressures.

 Enclosing of operations and usage exhaust ventilation at the place of chemical

release are recommended. Wearing of protective clothing for the experimentation

are needed (New Jersey Department of Health and Senior Services, 2002).

 Sodium phosphate dibasic waste is not considered as hazardous waste under

Resource Conservation and Recovery Act (RCRA), 40 CFR 261. Waste should be

disposed of while observing all precautions in compliance to prevailing

municipal, state and federal laws and standards from local environment agencies

(New Jersey Department of Health and Senior Services, 2002).

 Magnesium Chloride Hexahydrate

 Magnesium chloride hexahydrate is toxic to cardiovascular system and upper

respiratory tract. Long exposure to the substance can make the cause organ

damage. The substance is also an irritatant to the mucous membranes and upper

respiratory system. Magnesium chloride hexahydrate is not a fire hazard and is

stable under normal temperatures and pressures.

 Splash goggles, lab coat, dust respirator, boots, gloves and a self-contained

breathing apparatus should be used while performing experiments with

magnesium chloride hexahydrate.

 Magnesium chloride hexahydrate waste is not considered as hazardous waste

under Resource Conservation and Recovery Act (RCRA), 40 CFR 261. Waste

should be disposed of while observing all precautions in compliance to prevailing

ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 17

municipal, state and federal laws and standards from local environment agencies

(ScienceLab.com, 2013).

 Sodium Sulphate

 Sodium sulphate gives off irritating fumes that causes irritation in the respiratory

tract. When ingested, it causes digestive tract irritation, nausea, vomiting,

abdominal pain and diarrhoea. It also causes eye redness, eye injuries, eye pain

and skin sensitization when spilt in the eye and skin (PubChem, 2015). Sodium

sulphate is not a fire hazard and is stable under normal temperatures and

pressures.

 Usage of process enclosures, local exhaust ventilation, or other engineering

controls to keep airborne levels below recommended exposure limits are

recommended. Wearing of proper protective equipment must be employed

(ScienceLab.com, 2013).

 Sodium sulphate waste is not considered as hazardous waste under Resource

Conservation and Recovery Act (RCRA), 40 CFR 261. Waste should be disposed

of while observing all precautions in compliance to prevailing municipal, state

and federal laws and standards from local environment agencies

(ScienceLab.com, 2013).

 Tris(hydroxymethyl)aminomethane

 Tris(hydroxymethyl)aminomethane is not considered to be hazardous, but may

cause skin, eye, gastrointestinal and respiratory irritation in mild exposures.

Tris(hydroxymethyl)aminomethane is not a fire hazard and is stable under normal

temperatures and pressures.

ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 18

 Appropriate exhaust ventilation must be present at places were dust may form.

Wearing of proper protective equipment must be employed.

 Tris(hydroxymethyl)aminomethane waste is not considered as hazardous waste

under Resource Conservation and Recovery Act (RCRA), 40 CFR 261. Waste

should be disposed of while observing all precautions in compliance to prevailing

municipal, state and federal laws and standards from local environment agencies

(Sigma Aldrich, 2014).

 Hydrochloric Acid

 Hydrochloric acid may induce tissue damage particularly on mucous membranes

of eyes, mouth and respiratory tract. Skin contact may produce burns. Inhalation

of the vapour and sprays may produce severe irritation of respiratory tract,

characterized by coughing, choking, or shortness of breath. Severe over-exposure

can result in death. Eye inflammation is characterized by redness, watering, and

itching. Skin inflammation from the substance is characterized by itching, scaling,

reddening, or, occasionally, blistering. The substance can be toxic to kidneys,

liver, mucous membranes, upper respiratory tract, skin, eyes, circulatory system

and teeth. Repeated or prolonged exposure to the substance can produce organ

damage. Repeated exposure to hydrochloric acid may produce general

deterioration of health by an accumulation in one or many human organs.

Hydrochloric acid is non-flammable and is stable under normal temperatures and

pressures.

 Provision of exhaust ventilation or other engineering controls must be employed

to keep the airborne concentrations of vapors below their respective threshold

 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 19

limit value. Eyewash stations and safety showers must be near to the work-station

location. Wearing of proper protective equipment must be done (ScienceLab.com,

2013).

 Hydrochloric acid should be carefully diluted with water and neutralized to be

disposed of through a licensed contractor in compliance to applicable federal/local

regulations (BDH, 2005). Hydrochloric acid can be discharged through the sewer

when neutralized to a pH of 5.5 to 9.0 (Environmental Health and Safety, n.d.).

 Electrospinning Process

 The electrospinning process can cause needle pricks during installation; burning,

toxicity, fire and asphyxiation in case of certain polymer blend spills; and

electrical shocks when working with high voltage.

 Current risk preventive measure include gloves, fumehood use, insulating

materials and minimal to no direct contact to the set-up in order to prevent major

organ damage.

 Polymer blends left from the syringe during the electrospinning processes should

not be disposed through the sewer system. Utilized solvents should be left under

the fume hood before cleaning. Disposal of polymer blend should be in

accordance to prevailing municipal, state and federal laws and standards from

local environment agencies (Sun, Teng, Zhang, Dou and Li, 2013).

RESEARCH DESIGN AND STATISTICAL TESTS

Fiber diameter will be randomly selected and measured from the PLA-Nanosilica-CA

nanofibers and will be expressed as the average measurement. One-way ANOVA will be
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 20

employed to determine the differences between means. Duncan’s Multiple Range Test will be

used to compare each ratio at 5% level of significance.

BIBLIOGRAPHY

Bao, C.L.M.; Teo, E.; Chong, M.; Liu, Y.; Choolani, M.; Chan, J. (2013). Advances in Bone
Tissue Engineering, Regenerative Medicine and Tissue Engineering. Retrieved from
http://www.intechopen.com/books/regenerative-medicine-and-tissue-engineering/advances-
in-bone-tissue-engineering
BDH (2005). Hydrochloric acid. Retrieved from http://kni.caltech.edu/facilities/msds/HCl.pdf
BDH (2006). Methanol. Retrieved from http://www.methanol.org/getattachment/b327897d-
cbdd-4c9a-abc3-43d5a1590ce6/NorthAm_SDS_English-(1).pdf.aspx
BDH (2006). Dichloromethane. Retrieved from http://feql.wsu.edu/MSDS/Dichloromethane.pdf
Caltech (2006). Material Safety Data Sheet, Methanol. Retrieved from
http://kni.caltech.edu/facilities/msds/methanol.pdf
Chavan, P.; Bahir, M.; Mene, R.; Mahabole, M.; Khairnar, R. (2009). Study of nanobiomaterial
hydroxyapatite in simulated body fluid: Formation and growth of apatite. Retrieved from
http://www.researchgate.net/publication/216682560_Study_of_nanobiomaterial_hydroxyapat
ite_in_simulated_body_fluid_Formation_and_growth_of_apatite
Environmental Health and Safety, n.d. (2000). Procedures for Chemical Waste Disposal.
Retrieved from http://www.ehs.cornell.edu/chem_lab_safety/07.waste.disposal.pdf
Eastman Chemical Company (2014). Safety Data Sheet. Retrieved from
http://ws.eastman.com/ProductCatalogApps/PageControllers/MSDS_PC.aspx?Product=7101
2683
Helebrant, A.; Jonasova, L.; Sanda, L. (2001). THE INFLUENCE OF SIMULATED BODY
FLUID COMPOSITION ON CARBONATED HYDROXYAPATITE FORMATION.
Retrieved from http://www.ceramics-silikaty.cz/2002/pdf/2002_01_009.pdf
Liu, W.; Yeh, Y-C.; Lipner, J.; Xie, J.; Sung, H-W.; Thomopoulos, S.; Xia, Y. (2011).
Enhancing the Stiffness of Electrospun Nanofiber Scaffolds with Controlled Surface Coating
and Mineralization. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144316/
MedLine Plus (2014). Bone Diseases. Retrieved from
http://www.nlm.nih.gov/medlineplus/bonediseases.html
Nanosil (n.d.). Nanosil® Organosol Nano Silica Dispersion MSDS. Retrieved from
http://www.nanosilasiapacific.com/MSDS%208030.pdf
National University of Singapore (2008). Activity or Experiment-Based Risk Assessment Form.
Retrieved from
https://www.google.com.ph/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 21

=8&ved=0ahUKEwiAl-
u0zqjJAhXLkJQKHcwgA5UQFgghMAE&url=http%3A%2F%2Fwww.bioeng.nus.edu.sg%
2Fnanobio%2Fsafety%2FRisk%2520assessment%2520-
%2520Electrospinning.doc&usg=AFQjCNEtu6PM_8KRpVTqAKZJsZZnm4W9HQ&sig2=
dlwVdxhM6-Now-FSUun0ow
NatureWorks LLC (2009). NatureWorks® Polylactide Resin. Retrieved from
http://downloads.makerbot.com/filament/PLA+MSDS.pdf
New Jersey Department of Health and Senior Services (2002). Sodium Phosphate Dibasic.
Retrieved from http://nj.gov/health/eoh/rtkweb/documents/fs/1723.pdf
Olowa, L.; Nuneza, O. (2013). Brine Shrimp Lethality Assay of the Ethanolic Extracts of Three
Selected Species of Medicinal Plants from Iligan City Philippines. Retrieved from
http://isca.in/IJBS/Archive/v2/i11/12.ISCA-IRJBS-2013-177.pdf
PubChem (2015). Sodium sulfate. Retrieved from
http://pubchem.ncbi.nlm.nih.gov/compound/sodium_sulfate#section=Top
Repreper Tech Co. (2012). PLA Material Safety Data Sheet. Retrieved from
http://3dfilaprint.com/wp-content/uploads/2013/08/Repraper-PLA-MSDS.pdf
ScienceLab.com, (2013). Cellulose acetate MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=956
ScienceLab.com, (2013). Hydrochloric acid MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=9924285
ScienceLab.com, (2013). Magnesium chloride hexahydrate MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=9927214
ScienceLab.com, (2013). Potassium chloride MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=9927402
ScienceLab.com, (2013). Sodium bicarbonate MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=9927258
ScienceLab.com, (2013). Sodium chloride MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=9927593
ScienceLab.com, (2013). Sodium sulfate anhydrous MSDS. Retrieved from
http://www.sciencelab.com/msds.php?msdsId=9927278
Sigma Aldrich (2014). Tris(hydroxymethyl)aminomethane. Retrieved from
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=PH&language
=en&productNumber=252859&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.si
gmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2F252859%3Flang%3Den
Sigma Aldrich (2015). Silica. Retrieved from
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=PH&language
 ELECTROSPUN PLA-NANOSILICA-CA NANOFIBERS AS BONE TISSUE SCAFFOLD 22

=en&productNumber=718483&brand=ALDRICH&PageToGoToURL=http%3A%2F%2Fw
ww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Faldrich%2F718483%3Flang%3Den
Sun, G.E.; Teng, H.; Zhang, C.; Dou, Y.; Li, Y. (2013). Assembly of electrospinning setup for
spinning of nonwoven fibrous membrane- Basic Setup. Retrieved from
http://electrospintech.com/SOP-ES1001.html#.VlQe_XYrLIU
Tas, A.C. (1999). Synthesis of biomimetic CA-hydroxyapatite powders at 37°C in synthetic
body fluids. Retrieved from http://www.cuneyttas.com/hasbf37.pdf
TOXNET (2014). Dichloromethane. Retrieved from http://toxnet.nlm.nih.gov/cgi-
bin/sis/search/a?dbs+hsdb%3A%40term+%40DOCNO+66
Velleman NV (n.d.). Material Safety Data Sheet – PLA. Retrieved from
http://www.jameco.com/Jameco/Products/ProdDS/2184667.PDF
Wang, J.; Kalfas, I. (2015). Bone Grafts: New Developments. Retrieved from
http://www.spineuniverse.com/exams-tests/bone-grafts-new-developments
Ward’s Science (2008). Brine Shrimp. Retrieved from
https://media.vwr.com/emdocs/docs/scied/Brine_Shrimp.pdf

Yeum, J.H.; Park, J.H.; Kim, I.K.; Cheong, I.W. (2011). Electrospinning Fabrication and
Characterization of Water Soluble Polymer/Montmorillonite/Silver Nanocomposite Nanofibers
out of Aqueous Solution. Retrieved from http://cdn.intechopen.com/pdfs-wm/15416.pdf
Zhang, Y.; Venugopal, J.; El-Turki, A.; Ramakrishna, S.; Su, B.; Lim, C.T. (2008). Electrospun
biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue
engineering. Retrieved from
http://www.researchgate.net/publication/23182846_Electrospun_biomimetic_nanocomposite
_nanofibers_of_hydroxyapatitechitosan_for_bone_tissue_engineering