National Capital Region

Schools Division Office of Navotas City

Navotas National Science High School

Fabrication and Evaluation of Oyster Shell-derived Hydroxyapatite-based

Dental Resin Composite

In partial fulfillment of the requirements for the subject

Inquiries, Investigation, and Immersion

Name of Members:

Alejandrino, Sofia Loren Celine G.

Aviles, Anika Mae P.

Cruz, Ronalyn P.

De Jesus, Gian Brian J.

Gagatiga, Charlene Mae O.

Mr. Don King Evangelista Mr. John Kenneth Cruz

Research Advisers

2023

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1. INTRODUCTION

Oysters are bivalve mollusk types that grow in brisk water and can be naturally found in

coastal areas. Oysters are known for being well-balanced in maintaining their nutritional value,

being high in protein and minerals and low in fat, calories, and cholesterol, making them the top

choice for many consumers. Despite being one of the Southeast Asian countries with the highest

oyster aquaculture production, the recycling of waste shells in the Philippines is not well

developed, where it is typically discarded as food waste on land and in bodies of water

(Chilakala et al., 2019). On-land disposal of oyster shells causes microbial decomposition,

producing poisonous gasses such as ammonia (NH3) and hydrogen sulfide (H2S). Meanwhile,

soiling and infection of aquatic species brought on by oyster shells thrown on bodies of water

cause significant health hazards such as dengue, diarrhea, and malaria (Ruslan et al., 2021). Due

to these consequences, there is a need to repurpose oyster shells, and the typical method is to use

their high calcium carbonate (CaCO3) content.

Many studies have used calcium carbonate from various sources, including eggshells

(Azis et al., 2018), fish bones (Pal et al., 2017), and oyster shells (Verwilghen et al., 2009) to

produce hydroxyapatite. Hydroxyapatite (HA) is a natural mineral form of calcium apatite with

the chemical formula of Ca5(PO4)3(OH), usually written as Ca10(PO4)6(OH)2 to indicate that the

crystal unit cell is composed of two entities (Saeed et al., 2020). It is found inside the bones and

teeth of our body, making it rigid and robust. According to Nayak (2010), HA is the most often

used bioceramics among the various types in orthopedics and dentistry due to its non-toxic,

biocompatible, and osteoconductive characteristics. In dentistry, hydroxyapatite is used in dental

tissue regeneration, demineralization of enamel, and dental drug delivery by combining it with

other chemicals to form a biocomposite material.

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 On the other hand, Oral disease affects over 3.9 billion people globally, with dental

caries, gum disease, and oral malignancies affecting nearly half of the world's population (Kisley

et al., 2018) & (Peres et al., 2019). Severe periodontal (gum) disease and untreated tooth decay

in milk teeth are among the most common oral disorders worldwide. Therefore, filling is used to

treat a small hole, or cavity, in a tooth. Dentists remove the decayed tooth tissue and then fill the

space with a filling material (Dental Fillings, 2023). Dental filling can help to even out the

tooth's surface and improve jaw function for biting and chewing. Many patients with tooth decay

also have tooth sensitivity due to enamel loss, which improves with dental filling (Smith, 2019).

Numerous dental filling materials are available today, including gold, ceramic or porcelain, silver

amalgam, glass ionomer, and composite resin fillings (WebMD Editorial Contributors & Frisbee,

2021). Composite fillings are extensively utilized for dental fillings today because their different

fillers can match the original color and feel of the patient's teeth (Rangreez et al., 2019). The

three main parts of resin composite are polymer organic matrix, inorganic fillers, and initiator

system; hydroxyapatite, being the main mineral component of hard dental tissues, is responsible

for their hardness and other mechanical properties (Lezaja et al., 2013). Currently, most of the

HA materials used in biomedical applications are synthesized chemically, which is reported to be

expensive due to the cost of raw materials despite having lower quality than naturally-derived

HA (Razali et al., 2018).

To address the environmental and health effects caused by oyster shell waste, the

researchers of this study proposed to utilize natural HA from oyster shells to fabricate a dental

resin composite. With this aim, the mechanical properties of dental resin composite with

different amounts of HA as a filler were evaluated. This study will be beneficial to the dental

industry, proving that natural hydroxyapatite improves the mechanical properties of dental

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composite resin, resulting in oyster shells serving as a potential biomaterial source. Moreover, it

would also reduce the risks brought by the other types of dental filling and serve as an alternative

for high-cost raw materials.

2. METHODS

2.1. Materials

Sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium carbonate (Na2CO3),

ammonium phosphate ((NH4)3PO4), and deionized water, were purchased from ANSCOM

Medical Center at Bambang Street, Santa Cruz, City of Manila. Distilled water was purchased

from Mercury Drug-Navotas.

Duracryl Self Cure Denture Base Powder Clear and Self Cure Liquid were purchased

from New Citizen's Dental Supply at Quiapo, City of Manila.

Five kilograms of waste oyster shells were obtained from Malabon Public Market.

Magallana bilineata is the species of the collected oyster shell, as identified by the Institute of

Biology, College of Science at the University of the Philippines-Diliman.

2.2. Preparation of Powdered Oyster Shells

The collected shells were washed, brushed, and rinsed using distilled water. Then,

samples were obtained after discarding the unwell samples (shells with non removable dirt) and

removing the attached barnacles. After that, a powdered sample was obtained after the shells

were crushed and ground through a mortar and pestle. The obtained powder was sent to the

Department of Science and Technology - Industrial Technology Development Institute

(DOST-ITDI) for calcination at 500°C for 3 hours. After that process, the calcined powdered

samples yielded 1.705 kg.

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 Figure 1. Powdered oyster shells Figure 2. Powdered oyster shells after calcination
2.3. Synthesis of Oyster Shell-derived HA

2.3.1. Extraction of Calcium Carbonate from Oyster Shells

After preparing 1 L of 1 M NaOH in a 1 L beaker, 250 g of the samples was

added to it, and the formed mixture was stirred for 2 hours. The mixture was filtered and

washed with deionized water until the solution was neutral. Then, HCl was added and the

resulting mixture was stirred for 2 hours. The solution was filtered then the filtrate was

collected, separating the CaCl2 solution. Then, the obtained CaCl2 solution and Na2CO3

were mixed at a ratio of 1:1. The resulting solution was stirred rapidly for 30 seconds.

The particles of the solution were settled and filtered, then oven dried for 6 hours at

100°C to obtain CaCO3 powder (Lin et al., 2020).

Figure 3. Preparation of Figure 4. Mixed Powdered Figure 5. Separation of

NaOH Oyster Shell and NaOH solution
2.3.2. Microscopic Observation using Scanning Electron Microscope

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 The obtained powder was observed using a Hitachi SU 1510 Scanning Electron

Microscope (SEM) at the University of the Philippines-Manila to observe its morphology

and particle size.

2.3.3. Transforming Calcium Carbonate to Hydroxyapatite

20 g of obtained CaCO3 was added to a 250 ml beaker holding approximately

13.89 g of (NH4)3PO4 dissolved in 50 ml of distilled water. The suspension was stirred in

an air environment at 60°C, wherein the development of CO2 gas was observed. The

reaction continued for 72 hours. Then, fine powder was obtained from the white solids

via filtering. After that, the product was washed with distilled water to remove any

unreacted phosphate (Verwilghen et al., 2009).

Figure 6. Mixed CaCO3 and (NH4)3PO4 Figure 7. Oven-dried HA powder

solution
2.3.4. Characterization of Synthesized HA

The obtained powder was observed using a Hitachi SU 1510 SEM at the

University of the Philippines-Manila to observe its morphology and average size. The

proponents also conducted an X-ray Diffraction (XRD) Analysis to characterize the phase

composition, measurement of purity, and degree of crystallinity of the produced

hydroxyapatite at the Department of Science and Technology - Philippine Nuclear

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 Research Institute.

2.4. Fabrication of Dental Composite

To fabricate a biocomposite, two phases are needed: organic and inorganic. The organic

phase refers to the polymer matrices that undergo polymerization with the assistance of an

initiator and accelerator, while the inorganic phase consists of the fillers. In this research,

samples were prepared by mixing the acrylic-based resin (the monomer matrix) and oyster

shell-derived hydroxyapatite (filler). Various fractions of HA were added into the acrylic-based

resin, as observed in Table 1.

Table 1. Amount of resin and added HA into composite groups

2.5. Evaluation of the Mechanical Properties of HA-based Composite

Compression test is a mechanical test performed for composite materials. It measures the

material's behavior after being subjected to a compressive load at a relatively low and uniform

rate. The specimens were prepared in rectangular bars with a length of 50.8 mm and width and

thickness of 12.7 mm. After that, it was sent to the Physical and Performance Testing Laboratory,

Standards and Testing Division of the DOST-ITDI. Using the ASTM D695 method, the

specimen is placed between compressive plates parallel to the surface then the specimen is

compressed at a uniform rate. The maximum load is recorded along with stress-strain data while

an extensometer attached to the front of the fixture is used to determine modulus (Compression

Properties ASTM D695, ISO 604, n.d.). Overall, the specimen's compressive properties such as

compressive strength, stress at yield, and modulus were tested.

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3. RESULTS AND DISCUSSION

3.1. Preparation of Oyster Shells

The proponents obtained a 1744.90 g of powdered oyster shells as expressed in Table 2.

On the first day, five kilograms of oyster shells were cleaned and 474.82 g of it was initially

crushed into 297.78 g of powdered samples. While on the second day, the remaining oyster shells

were crushed into 1447.22 g powdered samples.

Table 2. Preparation of powdered oyster shells

Then, the researchers sent the sample to the Department of Science and Technology -

Industrial Technology Development Institute (DOST-ITDI) for calcination at 500° for 3 hours.

After that process, the calcined powdered samples yielded 1.7 kg.

3.2. Synthesis of Oyster Shell-derived HA

3.2.1. Preparation of CaCO3

The proponents obtained 215.49 g of CaCO3 after conducting two processes,

purification and reproduction. The purification process involves alkali and acid treatment,

where 527 g of sample was added to 1000 mL of NaOH and the product was rinsed using

450 mL HCl. After that, the obtained 400 mL of CaCl2 solution was used to produce

CaCO3 powder through the addition of 400 g Na2CO3.

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 Table 3. Extraction of CaCO3 from oyster shell powder

3.2.2. Morphology of CaCO3 using SEM

The obtained CaCO3 powder was observed under a Scanning Electron

Microscope (model Hitachi SU 1510) operating at a voltage of 10.0 kV. The sample got

burned, and there were no coatings to protect the sample, therefore the viewed details are

not visible. Still, the researchers observed that the CaCO3 particles were

irregularly-shaped with round edges and that the size of each particle varies in Figure 8

and Figure 9. Meanwhile, the sizes of the observed particles were 567 nm, 802 nm, and

1.55 um, as expressed in Figure 9.

Figure 8. SEM images of CaCO3 Figure 9. SEM images of CaCO3 with size
3.2.3. Synthesis of HA

Hydroxyapatite was synthesized using wet precipitation, as it is suitable for an

industrial production of HA since the only by-product is water (Anandan et al., 2023). As

such, 60 g of CaCO3 powder was added to a solution composed of 150 mL of 60°C

distilled water and 41.67 g of ammonium phosphate [(NH4)3PO4]. The suspension was

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degassed by continuously stirring every hour for 5 minutes. The pH level of distilled

water with ammonium phosphate started from 8.5, where it increased to 10.5 due to the

addition of ammonium phosphate. This change in pH level signifies that a reaction has

occurred. After 72 hrs, the precipitates were oven-dried at 100 degrees for 2-3 hours. The

obtained solids were thoroughly ground using a pestle and mortar and then examined for

phase purity and chemical composition using SEM and XRD.

3.2.4. Characterization of synthesized HA

The obtained CaCO3 powder was observed using Hitachi SU 1510 SEM on a

magnification of 30 and 50 um with a voltage of 15.0 kV. As observed in Figure 10, the

HA particles were irregularly-shaped with rough edges while the size of each particle

varies. Meanwhile, the sizes of the observed particles were 5.30 and 1.19 um, as

expressed in Figure 11.

Figure 10. SEM images of HA Figure 11. SEM images of HA with size

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