CHAPTER I

THE INTRODUCTION

This chapter states the reason of the researcher for pursuing their topic and the

problems that will be answered during the process of the experiments. The hypotheses,

both null and alternative are also included, together with the rationale of the study. In this

chapter, the scope and the limitations of the study are read including the related studies

and literature that the researcher used as a handy tool in the completion of the study.

Background of the Study

Surgical sutures are staple instruments in the field of medicine and is by far the

most common. Often utilized to prevent bacterial infection, stop bleeding wounds

instigated by injuries, and reduce acute scarring, the technique involving suturing dates

back into Ancient Egypt circa 30,000 years ago. As medical practitioners since then have

continued to advance their work, a wide array of materials have been used to produce

different types of sutures, that of which includes naturally found constituents such as silk,

cotton, linen, and catgut, all of which depending on what specific part of the body the

suture is going to be used. Synthetic materials have also been used to produce

commercially available sutures and is, in fact, still used in modern medicine, one of

which is polymer. (Struszczyk, 2002)

Polymer based sutures are known to satisfy most of the physical (sterility, tensile

strength, uniformity in diameter, and pliability) and chemical properties a medical suture

presents. Although still not ideal, it has gained its immense attraction from scientists and

1
health practitioners alike due to its carbon-based chemistry which is considerably closer

to biological tissue than inorganic materials. (Maitz, 2015)

It is in this regard that a type of naturally occurring polymer found in the shells of

the Metapenaeus ensis, common name Greasyback Shrimp or Suahe, endemic to the

Philippine seawaters, called chitosan is being viewed of the researcher of the study as a

potential non-synthetic material to be used for medical sutures. A chitosan-based suture

presents a low-inflammatory response material and is significantly much less painful to

remove as opposed to commercially-made sutures (Montenegro & Godeiro, 2012) as it

shows a biological aptitude to stimulate cell proliferation and tissue organization. It can

also play the role of a biological primer for cell-tissue proliferation and reconstruction.

(Muzzarelli et. al, 1988)

Statement of the Problem

General Problem

This study aims to utilize chitosan fibers from M. ensis (Greasyback

Shrimp) shells to create a medical suture and to test the chitosan’s feasibility as a

main constituent in the medical suture.

Specific Problems

Specifically, this study attempts to answer the following problems:

1. Will chitin be extracted from M. ensis (Greasyback Shrimp) shells

through deproteinization and demineralization?

2
 2. Will the chitin be converted into chitosan fibers through Interfacial

Polyelectrolyte Complexation?

3. Will there be a difference in the fiber produced if the shells of M. ensis

(Greasyback Shrimps) are treated with a higher concertation of

chemicals during the processes deproteinization and demineralization?

 Will there be a significant difference in the chitosan-derived

surgical sutures and the commercially available suture in terms

of tensile strength? diameter? sterility? pliability?

Hypotheses

The researcher came up with possible hypotheses to the problems that might

encounter in the study, Chitosan Fibers from M. ensis (Greasyback Shrimp) shells as

Medical Suture. Below are the researcher’ hypotheses in null and alternative form.

Null

1. Chitosan fibers from M. ensis (Greasyback Shrimp) shells cannot create a

medical suture and the chitosan is not feasible as a main constituent for

medical suture.

2. Chitin cannot be extracted from M. ensis (Greasyback Shrimp) shells through

deproteinization and demineralization.

3. Chitin cannot be converted into chitosan fibers through Interfacial

Polyelectrolyte Complexation.

3
 4. There is no difference with the fibers produced when shells of M. ensis

(Greasyback Shrimps) were treated with a higher concentration of chemicals

in the processes of deproteinization and demineralization.

5. There is no significant difference in the chitosan-derived surgical sutures and

the commercially available suture in terms of its diameter.

Alternative

1. Chitosan fibers from M. ensis (Greasyback Shrimp) shells can create a

medical suture and the chitosan is feasible as a main constituent for medical

suture.

2. Chitin can be extracted from M. ensis (Greasyback Shrimp) shells through

deproteinization and demineralization.

3. Chitin can be converted into chitosan fibers through the Interfacial

Polyelectrolyte Complexation.

4. There is a difference with the fibers produced when shells of M. ensis were

treated with a higher concentration of chemicals in the processes of

deproteinization and demineralization.

5. There is a significant difference in the chitosan-derived surgical sutures and

the commercially available suture in terms of its diameter.

Theoretical Framework

A pivotal theory which underpins this study is George Winter’s (1962) Moist

Wound Healing Theory. While the central concern of this thesis is with regard to chitosan

4
effectiveness, the foundations of the study would be nowhere found without its anchorage

on healing theory—a component which serves as one of the objectives sought by the

researcher. In order to better understand surgical methods and their instruments thereof, it

is of great import to refer to propositions which have laid the groundwork to their

perfection, that is, their healing or curing properties.

Chitosan is reputable in its own right, yet to test its viability as an added substance

to the creation of a surgical suture, the fundamentals of wound dressing must be

addressed. The aforementioned premise thus begs the researcher to seek a salient theory

therein which has fostered immense succor to the field of surgical science—the Moist

Wound Healing Theory. According to this theory by Winter (1962 as cited in Bryan,

2004) epithelialisation occurred twice as fast for wounds kept moist as opposed to dry

wounds. The healing optimization afforded by the moist environment not only aids

significantly towards the betterment of scarred patients, but also to the application of their

surgical dressings thereof. Moreover, Winter also affirms (1962 as cited in Bryan, 2004)

that the incidence of a wound being infected would decrease if the surgical process would

be conducted in this ideal type of atmosphere. Such finding has thus been groundbreaking

in medical field and practice as it would expand the available literature and knowledge on

wound dressing and healing. This theory has also been backed up by numerous

researcher, most importantly Hinman and Maibach (1963 as cited in Bryan, 2004) who

have tested the theory on humans; as it must be noted that Winter has only examined his

theory preliminarily on pigs. As the researcher of this thesis shall test the effectiveness of

the surgical suture, this theory will be of great assistance having borne in mind and

5
practice, for the dryness or moistness of the surrounding environment can be a factor in

the efficacy of Chitosan-based surgical sutures.

On another note, Tachibana et al.’s (1988) theory of Chitin suture strength

reinforces a primary objective by the researcher, namely, to prove the effectiveness of the

surgical suture infused with fibers from the said substance. According to them, the

properties possessed by a Chitin-based suture made it a viable choice as a surgical aid

and instrument (Tachibana et al., 1988). For one, its smaller elongation capacity equates

to a lower probability of loosening once applied to wounds. Another advantage it affords

is its tensile strength which spells for a secure and sturdy implement for wound dressing.

Ultimately, Tachibana et al.’s research has led them to conclude that “Chitin is a suture

material which is easily ligated and difficult to loosen” (1988, p. 538), thus fortifying the

bedrock of this thesis.

Conceptual Framework

The researcher gathered their data from M. enensis (Greasyback Shrimp), which

was commercially available and was easily bought from local markets. The researcher

proceeded into the execution of the set of methods that have been deemed necessary to

extract the chitosan from the M. ensis (Greasyback Shrimp) shells namely, (a) manual

head and carapace separation via Raw Material Preparation, (b) Chitin Extraction through

deproteinization and demineralization, and lastly the (c) Interfacial Polyelectrolyte

Complexation which resulted in the needed chitosan-based sutures. The data that were

6
gathered via the methods specified by the researcher were used to attempt to answer the

specific problems brought forward in the earlier parts of the study.

OUTPUT

Chitosan fibers from

INPUT
Chitosan fibers from
M. ensis (Greasyback
Shrimp) shells
PROCESS
The researchers will
follow the set of pre-
determined methods in
order to come up with
the chitosan-based
suture and determine
the its feasibility,
properties, and
significant difference
through a series of
tests.
M. ensis (Greasyback
Shrimp) shells can
create a medical suture
and is feasible as a
main constituent in the
medical suture.
---------or---------
Chitosan fibers from
M. ensis (Greasyback
Shrimp) shells cannot
create a medical suture
and is not feasible as a
main constituent in the
medical suture.

Significance of the Study

The notable significance of the availability of medical tools in the field of

medicine has been proved countless of times along with the modernization of human

civilization. Progressions in the medical field have provided humanity with better

solutions to answer the most confounding cases with regards to the body through the aid

of science and research. In attempting to create a medical suture out of a naturally

occurring biopolymer which is chitosan, the researcher of the study were not only aiming

7
to find benefit for themselves but also for the medical community, the government branch

which deals with the citizens’ health-related concerns (Department of Health), and the

country itself. Additional sufficient knowledge was quite enough for the researches of the

study to properly execute the creation of the expected product. The set of procedures

enabled them to observe the outcome of their research which allowed them to properly

reach their inferences and conclusions before, during, and after the study was conducted.

Patients and medical practitioners alike will benefit from the study as well since

the actual testing and usage of the resulting product will be utilized by these people. The

response that was acquired have are going to be crucial in acquiring most of the vital

information the researcher of the study will use. This will include answers to the

problems posed in the earlier parts of the study.

Providing cheap alternatives without comprising quality is one of a government

institution’s goal in responsibly taking care of a certain country’s citizens. The

Department of Health (DoH) is one of these government institutions. Feasibility of the

expected product provides them another option when dealing with the distribution of safe

and efficient medical tools to the general masses.

Achieving a goal this significant will also benefit the country in a major scale. It

will then be provided with an innovative tool that will be able to exhibit the properties of

commercially made sutures that will potentially aid in providing better results in terms of

wound healing and recovery.

Scope and Delimitations of the Study

8
 The study’s purpose is to utilize chitosan fibers from M. ensis (Greasyback Shrimp)

shells as medical sutures.

Chitosan is a natural biopolymer abundant from different sources like crabs and

lobster shells, insects, and fungi. This study, however, only utilized one source of

chitosan and that was from shrimp. The researcher of the study also limited the source

further into only one specific specie of shrimp, M. ensis (Greasyback Shrimp as it is one

of the most common types that can be found in the Philippine setting. The researcher was

not able to test the suture on a live organism as time constraints and possible additional

requirements with regards to animal and/or human welfare must be met and

accomplished. In relation with animal and/or human welfare, the researcher was only able

to test whether chitosan was a feasible medical suture constituent and not whether the

resulting fiber was an ideal suture that was able to satisfy all types of wounds in the

different parts of the body.

9
Definition of Key Terms

 Biopolymer (noun)

o a polymeric substance (such as a protein or polysaccharide) formed in a

biological system

 Carapace (noun)

o a bony or chitinous case or shield covering the back or part of the back of

an animal (such as a turtle or crab)

 Catgut (noun)

o a tough cord made usually from sheep intestines

 Chitin (noun)

o a horny polysaccharide that forms part of the hard outer integument

especially of insects, arachnids, and crustaceans

 Chitosan (noun)

o a substance formed from chitin by partial deacetylation with alkali

 Crustacean (noun)

o any of a large class (Crustacea) of mostly aquatic mandibulate arthropods

that have a chitinous or calcareous and chitinous exoskeleton, a pair of

often much modified appendages on each segment, and two pairs of

antennae and that include the lobsters, shrimp, crabs, wood lice, water

fleas, and barnacles

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 Interfacial Polyelectrolyte Complexation (noun)

o a type of step-growth polymerization in which polymerization occurs at an

interface between an aqueous solution containing one monomer and an

organic solution containing a second monomer

 Litigate (intransitive verb)

o to carry on a legal contest by judicial process

 Polymer (noun)

o a chemical compound or mixture of compounds formed by polymerization

and consisting essentially of repeating structural units

 Shrimp (noun)

o any of numerous mostly small and marine decapod crustaceans (suborders

Dendrobranchiata and Pleocyemata) having a slender elongated body, a

compressed abdomen, and a long spiny rostrum and including some

(especially family Penaeidae) that are commercially important as food;

also : a small crustacean (such as an amphipod or a branchiopod)

resembling the true shrimp

 Suture (noun)

o a stitch made with a suture

o a strand or fiber used to sew parts of the living body

o the act or process of sewing with sutures

11
 CHAPTER II

REVIEW OF RELATED LITERATURE

The researcher has gathered legal bases, literature, and other studies that had

comparable similarities with the study they are conducting. The resources come from

both local and foreign sources. All the information they gathered served as the basis of

their study and aided the understanding their study in a deeper sense and are presented in

this chapter.

Related Readings

A. Foreign

According to Section 878 of the Code of Federal Regulations Title 21, medical

devices are classified into Class I, II and III and regulatory requirements and controls are

defined by these classifications. The regulatory control increases from Class I to III.

Absorbable sutures such as polyglycolide and poly(l-lactide) sutures are under Class II

and these devices require general and special controls. General controls consist of pre-

market notification or the “510(k) Program”, properly labeling of the medical device, and

compliance with the U.S. Food and Drug Administration’s quality system regulations.

12
Special controls for these devices require guidelines in the application, usage and

handling of the device, special labeling, and a guidance document.

B. Local

According to Section 4 of the Republic Act No. 8503 entitled “Health Research

and Development of 1998”, the National Institute of Health is established to promote

science, technology research, and development in the field of health. Innovative ideas are

encouraged to enhance current medical techniques, medical equipment and medical drugs

to ensure continuous development. For the reason that it will help attain self-sufficiency

and global competitiveness in the manufacturing of health products and devices.

Related Literature

In 2007, Singh and Ray in their article on the Journal of Macromolecular Science

said that chitosan, derived from the second most abundant organic resource known as

chitin, has advantages in biomedical applications, especially in manufacturing absorbable

sutures, with its properties such as nontoxicity and biodegradability. This polymer is a

biocompatible material that disintegrates into harmless products such as amino sugars,

which can be eventually absorbed completely by the body. Sutures made from chitin,

which is the source of chitosan, is used specially when in contact with bile, urine, and

pancreatic juices, which are problem areas with other sutures. In addition, one of the

potentials of chitosan in biomedical applications is that it has also been claimed to

accelerate and aid in wound-healing process. An investigation on the possibility of

chitosan as wound healing accelerator done by Malette, Quigly, and Adickes show that

no abnormal tissue reactions or infections were observed in using chitin sutures.

13
 Montenegro and Godeiro mentioned in their article entitled “Chitosan Based

Suture – Focusing on the Real Advantages of an Outstanding Biomaterial” that

monofilament fibers are more suitable especially in closing wounds because unlike

multifilament sutures, they do not produce a capillary effect that could transport

microorganisms from one wound to another. For this reason, chitosan has antimicrobial

properties and does not elicit a high inflammatory response as it can be manufactured as a

monofilament fiber with required mechanical properties. The need for removal of sutures

is not a necessity for a biodegradable suture but their degradation time undefined. In

other cases, removal of suture even with biodegradable ones is needed because

complications may occur. With chitosan sutures, applying an acidic solution for few

hours dissolves the suture from the skin instead of in vitro removal, which is a pain-free

method.

Related Studies

In a study conducted by Rivelino Montenegro and Thomas Freier, chitosan has

been suggested as an acceptable candidate for bioabsorbable surgical sutures because of

its high biocompatibility. The chitosan fibers’ biocompatibility was tested by implanting

the suture subcutaneously in the neck of the mice that served as the sample. Then, weeks

after, the suture was removed and the area of implantation was examined histologically.

There are no reported signs of inflammation or other significant changes. The result

shows that chitosan is compatible with the living tissue of the organism. On the other

hand, a research led by Kyung-Hye Jung in 2007 suggests the possible anti-bacterial

propertyof PET/chitosan nanofibrous mats. The polymers of Polyethylene terephthalate

14
(PET) and chitosan were mixed through the process called electrospinning technique on

to the PET micro-nonwoven mat to be used for biomedical applications. The nanofibers

of PET/chitosan were distributed evenly on its surface with a diameter of 500 and 800

nm. The antibacterial activity of the PET/chitosan nanofiber mats were tested by

evaluating its inhibitory property towards Staphylococcus aureus and Klebsiella

pneumoniae. The result of the evaluation showed a significantly higher growth inhibition

rate of the PET/chitosan nanofiber mats compared to the PET nanofiber mats. Aside from

this, the addition of chitosan into the mat enhanced the wettability of the PET nanofibers.

Lastly, the PET/chitosan nanofibers mats shows better tissue compatibility than PET

nanofibers mats. Lastly, a study led by Mi-Sun Kim in 2002 shed light regarding the

inhibitory and anti-inflammatory attributes of water-soluble chitosan. A chronic

inflammatory response between β-amyloid (Aβ) and interleukin- 1β (IL-1β) is

responsible for the pathology of Alzheimer’s disease. The cytotoxicity was evaluated to

test the biological effect of water-soluble chitosan production of pro-inflammatory

cytokine and inducible nitric-oxide synthase (iNOS) in human astrocytoma cells

galvanize with Aβ and IL-1β particles. The expression of iNOS stimulated with Aβ and

IL-1β and was partially regulated by the usage of water-soluble chitosan. The result

shows the regulatory effects of water-soluble chitosan to human astrocytoma cells. In line

with this, anti-inflammatory property of water-soluble chitosan was exhibited and this

research proved that water-soluble chitosan may reduce and delay Alzheimer’s disease

pathological events.

15
 CHAPTER III

MATERIALS AND METHODS

In this chapter, the researcher lays out the procedures regarding their study,

Chitosan Fibers from M. ensis (Greasyback Shrimp) Shells as Medical Sutures. To

achieve that endeavor, the researcher focused on the materials that they used in order to

perform the experiment needed in the study, as well as the particular procedure. The data

gathering procedure is also viewed from this chapter together with the statistical

treatment the researcher used in order to compare the control and experimental groups.

Research Design

The researcher used the single group experimental design which involved one (1)

control group (commercially made medical suture) and one (1) experimental group

(chitosan-derived suture). The experimental group was tested, observed, and compared

with the control group. This provided the answers essential to the study as the effectivity

16
of the chitosan derived suture tell whether it can be an ideal replacement for

commercially made sutures.

Materials

 Shrimp Shells (10g)  Sodium Alginate

 Hydrochloric Acid (HCl)  Acetic Acid

 Sodium Hydroxide (NaOH)  Deionized Water

 Distilled Water

Figure 1. Shrimp shells (10g) Figure 2. Hydrochloric Acid

17
 Figure 3. Sodium alginate Figure 4. Acetic acid

Figure 5. Sodium Hydroxide

Figure 6. Deionized Water Figure 7. Distilled Water

Instruments

18
 Mortar  Vials

 Pestle  Petri Dish

 Curved Tip Forceps  Stirring Rod

 Beakers  Heater

 Graduated Cylinder  Caliper

 Dropper  Digital Weighing Scale

 Oven

Figure 8. Mortar and Pestle Figure 9. Curved Tip Forceps

Figure 10. Beakers Figure 11. Graduated Cylinder

19
 Figure 12. Dropper Figure 13. Oven

Figure 14. Vial Figure 15. Petri Dish

Figure 16. Stirring rod Figure 17. Heater

20
 Figure 18. Vernier Caliper Figure 19. Digital Weighing Scale

Procedure

Raw Material Sample Preparation

1. Shells of M. ensis (Greasyback Shrimp) were obtained from commercially

available shrimps of the same specie in a local market.

Figure 20. Procurement of M. ensis

2. The shells were washed, air dried, and refrigerated for twenty-four (24) hours.

21
Figure 21. Washing of the M. ensis shells Figure 22. M. ensis shells soaked in water

Figure 23. Drying of M.

ensis shells

3. The samples were then oven dried for two (2) consecutive days at 65° Celsius

for one (1) minute.

Figure 24. Dried M. ensis shells

22
 4. The total amount of M. ensis shrimp shells gathered accumulated into ten (10)

grams. Two (2) set-ups were made with an equal amount of samples (5 g of

shells each) and were aptly named Set-Up A and Set-Up B.

Figure 25. 5 g of dried M. ensis shells were weighed for Setup A and Setup B

Chitin Extraction (Deproteinization and Demineralization)

1. Set-Up A was diluted in 4% NaOH while Set-Up B was diluted in 8% NaOH both

at room temperature for twenty-four (24) hours.

Figure 26. 500mL of distilled water mixed with 20 g of NaOH for Setup A

23
 Figure 27. Setup A – shells of Figure 28. Setup B – shells of M. ensis
M. ensis treated in 4% NaOH treated in 8% NaOH
2. After the twenty-four (24) hours, both set-ups were repeatedly rinsed with

distilled water until the scent of the NaOH faded. This process caused the

deproteinization of the shells.

Figure 29. Draining of alkali from the shrimp shells

3. Immediately afterwards, the deproteinized shells of Set-Up A were treated with

4% HCl while Set-Up B were treated with 8% HCl both at room-temperature for

twelve (12) hours for demineralization.

24
Figure 30. Setup A with 4% HCl Figure 31. Setup B with 8% HCl

4. The acid was drained off from the chitin, washed with distilled water, and finally

dried at room temperature.

Figure 32. Air drying of shrimp

shells in Setup A Figure 33. Air drying of shrimp
shells in Setup B

5. The process was repeated for both set-ups with Set-Up A having 2% NaOH and

1% HCl and Set-Up B with 4%

NaOh and 2% HCl.

25
 Figure 34. Setup A with 2%
NaOH

Figure 35. Setup B with 4% NaOH

Conversion of Chitosan from Chitin

1. Both Set-Ups were deacetylated to form chitosan by treating with 65% NaOH for

three (3) days at room temperature.

Figure 36. 500ml of distilled water mixed with 325g NaOH

26
Figure 37. Setup A with 65% NaOH Figure 38. Setup B with 65% NaOH

2. After the three (3) days, the set-ups were drained and washed repeatedly with

distilled water until the scent of the NaOH faded.

Figure 39. Setup A washed with Figure 40. Setup B washed with
distilled water distilled water
3. The chitosan set-ups were further dried at room temperature and stored.

distilled water

Figure 41. Setup A washed with

27
 Figure 42. Setup B air drying

Figure 43. Setup A and B

Acetic Acid Test: Solubility of Chitosan

1. Chitosan dissolves completely in 1% acetic acid.

28
 Figure 44. 1% Acetic Acid

2. A few grams from both set-ups were obtained and doused with 1% acetic acid and

stirred continuously for 20 minutes.

Figure 45. Setup A mixed with Figure 46. Setup B with 1%

1% Acetic Acid Acetic Acid stirred for 20
minutes

Interfacial Polyelectrolyte Complexation

1. Chitosan and sodium alginate were employed. A 1% chitosan solution in 1%

acetic acid and 2% sodium alginate solution in deionized water were prepared.

29
Figure 47. Setup A – 1% chitosan Figure 48. Sodium alginate mixed w/
solution in 1% acetic acid deionized water

Figure 49. Setup B – 1% chitosan solution in 1% acetic acid

2. The solution interface was created by first placing droplets of the chitosan

solution and sodium alginate on top of each other.

3. The chitosan fibers were fabricated by drawing up the interface between two

oppositely charged polyelectrolyte solutions using curved tip forceps. The curved

tip forceps were used to bring the two (2) droplets in contact and the upward

30
 drawing motion was instantly commenced until one of the polyelectrolyte phases

is depleted.

Figure 50. Fiber being drawn Figure 51. Fiber was being drawn from
from setup A setup B

Statistical Treatment

The researcher used percentage difference to distinguish whether there is a

significant difference between the controlled variable (Commercially Available Suture;

Plain Catgut Suture) and the experimental variables (Set-Ups A and B). The researcher

used this formula:

|(Experimental – Controlled) / Control| x 100

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 CHAPTER IV
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

In this chapter, the data gathered from the experimentation proper are

presented, interpreted, and analyzed to be able to answer the specific problems

and the research objectives. This chapter also discusses the results and findings

the researcher obtained using the research instruments utilized by the researcher.

Furthermore, it also discusses the mechanical property, specifically thickness, of

the chitosan fiber which was formed through the conversion of the extracted

chitosan from the shells of M. ensis (Greasyback Shrimps) into fiber by Interfacial

Polyelectrolyte Complexation.

Presentation of Data

Table 1. The measured diameters of the threads in Setup A and Setup B

SETUP A SETUP B
Ratio of droplets of
Ratio of 1%Chitosan
1% Chitosan
Solution with 1%
Solution in 1% Diameter of the Diameter of the
Acetic Acid to 2%
Acetic Acid to 2% Thread (µm) Thread (µm)
Sodium Alginate
Sodium Alginate
Solution
Solution
3:1 10 µm 3:1 10 µm
3:2 20 µm 3:2 80 µm
3:3 40 µm 3:3 90 µm

32
The values in table 1 show the measured diameters of the threads in Setup A and Setup B

that were employed in the different ratio of droplets of 1% chitosan solution in 1% acetic

acid to 2% sodium alginate solution. Both Setup A and Setup B serve as the experimental

groups.

Photoset 1. Chitosan Fibers drawn through Interfacial Polyelectrolyte Complexation from Setup

A with ratio of droplets of 3:1, 3:2, and 3:3 respectively

33
Table 2. The significant difference in the measured diameters of the threads between Setup A and

the plain catgut suture

Ratio of droplets of

1% Chitosan Solution Diameter of the Diameter of the

in 1% Acetic Acid to Thread (µm) for Plain Catgut Result (%)

2% Sodium Alginate Setup A Suture (µm)

Solution
3:1 10 µm -92.3%
3:2 20 µm 130 µm -84.6%
3:3 40 µm -69.2%

The values in table 2 display the significant difference of the measured diameter

of the threads between Setup A and the plain catgut suture. There is a -92.3% significant

difference between the thread yielded from the solution with 3:1 ratio and the plain catgut

suture. On the other hand, the measured diameters of the threads between Setup A in 3:2

and 3:3 ratios and the plain catgut suture exhibit -84.6% and -69.2% significant

differences respectively.

34
Photoset 2.Chitosan Fibers drawn through Interfacial Polyelectrolyte Complexation from Setup

B with ratio of droplets of 3:1, 3:2, and 3:3 respectively

Table 3. The significant difference in the measured diameters of the threads between Setup B and

the plain catgut suture

Ratio of droplets of

1% Chitosan Solution Diameter of the Diameter of the

in 1% Acetic Acid to Thread (µm) for Plain Catgut Result (%)

2% Sodium Alginate Setup Suture (µm)

Solution
3:1 10 µm -92.3%
3:2 80 µm 130 µm -38.5%
3:3 90 µm -30.8%

The values in table 3 exhibit the significant difference of the measured diameter

of the threads between Setup B and the plain catgut suture. A significant difference of

-92.3% is obtained for comparing the measured diameter of the thread produced from the

35
ratio of 3:1 and the plain catgut suture. On the other hand, the measured diameters of the

threads between Setup B in 3:2 and 3:3 ratios and the plain catgut suture exhibit -38.5%

and -30.8% significant differences respectively.

Interpretation and Analysis of Data

Figure 52. Microscopic view of the Figure 53. Microscopic view of the
chitosan fiber chitosan fiber

Figure 54. Microscopic view of the

chitosan fiber

In both Setup A and Setup B, the researcher were able to obtain chitosan fibers

through the process of Interfacial Polyelectrolyte Complexation. The fibers were then

measured depending on the ratio of droplets of 1% chitosan solution in 1% acetic acid to

36
2% sodium alginate solution. The data acquired from both setups show a huge micro

difference among the diameters of the monofilament chitosan fibers that were produced.

Fibers procured from the 3:1 ratio have the same measure of the diameter. However,

fibers attained from the ratios 3:2 and 3:3 have an immense difference of 60 µm and 50

µm respectively. Setup B, which was treated using higher concentration of chemicals

during the process of deproteinization and demineralization, produced fibers that

comparably have a longer diameter as compared to fibers from Setup A. When compared

to the commercially available plain catgut suture that has a diameter of 130 µm, both

chitosan fibers from Setup A and Setup B exhibit a massive difference in the measured

diameter of the thread.

CHAPTER V

37
 SUMMARY, CONCLUSION, AND RECOMMENDATIONS

With the data gathered and interpreted, the researcher came up with the result of

the study “Chitosan Fibers from M. ensis (Greasyback Shrimp) Shells as Medical

Sutures”. This chapter also includes the recommendation and summary of the research.

Summary

The researcher fundamentally noted that the essence of the study revolved around

the creation of a chitosan based medical suture that could potentially be comparable to

the sutures that are commercially available. Through further analysis and execution of the

experimentation process underpinned by factual evidences and bases, the researcher were

able to extract the needed naturally occurring biopolymer, chitosan, from the shells of M.

ensis (Greasyback Shrimp) through deproteinization and demineralization. The

researcher were also able to conduct the solubility test through the usage of acetic acid

for both of their set-ups which provided them with enough knowledge to verify that they

have extracted chitosan. In addition, fibers were also efficaciously drawn from the

chitosan and sodium alginate solutions via the process of Interfacial Polyelectrolyte

Complexation.

Conclusion

Using the aforementioned processes (Chitosan extraction through deproteinization

and demineralization, solubility test, and Interfacial Polyelectrolyte Complexation) that

served as the backbone of the study, the data that were interpreted, analyzed and

evaluated lead the researcher to a conclusion, which accepts the alternative hypothesis

38
stated in Chapter I. Chitosan fibers from M. ensis (Greasyback Shrimp) shells can indeed

create and is a feasible main constituent in the medical suture.

To answer the specific questions posed in the former parts of the study, chitosan

fibers from M. ensis (Greasyback Shrimp) shells can create a medical suture and is a

feasible main constituent for medical suture. Specifically, chitin can be extracted from M.

ensis (Greasyback Shrimp) shells through deproteinization and demineralization. As

exhibited in Figures 27 and 28, the visible shells mixed into its solution almost

immediately. Initial observations made by the researcher suggest that after the shells

combined with the medium, the resulting solution became more viscous. This property

was exhibited more with the shells that came from Set-up B. This certain property may be

attributed to the fact that Set-up B contained shells of M. ensis (Greasyback Shrimp) that

were treated with a higher concentration of chemicals in the processes of deproteinization

and demineralization. The shells for Set-up B were also cut into smaller pieces before the

three-day exposure to sodium hydroxide. These extra measures were not done to Set-up

A, which yielded a less viscous chitosan solution.

Figures 32 and 33 show how chitin can be converted into chitosan fibers through

the Interfacial Polyelectrolyte Complexation. Fiber drawing requires different draw rates

to complement the viscosity of the chitosan solution. Since Set-up B of the study yielded

a more viscous solution, the researcher had to adjust the draw rates in order to attempt to

obtain a fiber that was of uniform diameter.

Tables 1, 2, and 3 of the study shows the difference of the diameters between (a)

the fibers drawn from Set-up A and Set-up B, (b) the fibers from Set-up A and the control

group, and (c) the fibers from Set-B and the control group, respectively. The most

39
prominent differences between the three are quite simple enough. Based on the data the

tables provide, Set-up A almost had the same diameters as Set-up B. When compared to

each other and with the commercially available suture, none of the two set-ups seem to be

up par with the commercially available suture based on diameter. The control group

spans a 130 µm while Set-ups A and B yielded sutures that were evidently smaller in

diameter. However, the possibility of a chitosan based suture isn’t entirely impossible. As

there is no ideal suture which can be used for every circumstance in the field, medical

practitioners have to use sutures that have varying diameters and materials used to create

them. The standard diameters brought forward by the US Pharmacopeia set a few

standards on the usage of the sutures. The commercially available suture the researcher

had was under USP 4-0 or 5-0 (diameter is approximately 0.13mm/130 µm) could be

used for large vessel repairs while the fibers the researcher were able to draw could be

under USP 11-0 (diameter is approximately around 0.01mm to 0.02mm/10-20 µm) and

may be used for ophthalmologic operations or microsurgical repairs.

Recommendations

Throughout the time that the study, “Chitosan Fibers from M. ensis (Greasyback

Shrimp) Shells as Medical Sutures” was conducted, it was apparent that there was a

multitude of setbacks the researcher have noticed even before the actual experimentation

began. For one, the proper equipments and/or machines that could prove to be useful in

the study weren’t readily available in the locale of the researcher. Equipments that could

automatically draw the chitosan fibers for the Interfacial Polyelectrolyte Complexation

was terribly needed as draw rates affect the outcome of the fiber and at least an autoclave

could have been present such that the sterilization of the tools used in the study could be

40
more precise and rounded. A short time frame to complete the study might have also

played a detrimental role in the outcome of the study. A few more set-ups with different

variables could give more comprehensive results and could be easily compared with one

another and with a control group. It is also recommended that the next researcher of the

study attempt to create a suture and test its specific mechanical properties and other

properties that could make the results more reliable.

41
 Bibliography

An Act Providing For The Promotion Of Health Research And Development,

Establishing For The Purpose The National Institutes Of Health (NIH), Defining
Its Objectives, Powers And Functions, And For Other Purposes, R.A. 8503, 10th
Cong. (1998). Retrieved from The LawPhil Project Databank.

Azuma, K., et al. (n.d.). Chitin, Chitosan, and Its Derivatives for Wound Healing: Old
and New Materials. doi:10.3390/jfb6010104

Hudson, S., et al. (2005): Hemostatic Agents Derived from Chitin and Chitosan, Journal
of Macromolecular Science, Part C: Polymer Reviews, 45:4, 309-323

Jung, K., et al. (2007). Preparation and antibacterial activity of PET/chitosan nanofibrous
mats using an electrospinning technique. Journal of Applied Polymer
Science, 105(5).

Kim, M., et al. (2002). Water-soluble chitosan inhibits the production of pro-
inflammatory cytokine in human astrocytoma cells activated by amyloid beta
peptide and interleukin-1beta. Neuroscience Letters., 105-109

Lim, C., & Halim, A. (2010). Biomedical-Grade Chitosan in Wound Management and Its
Biocompatibility In Vitro. Biopolymers.

Mahoney, C., et al. (2012) Nanofibrous Structure of Chitosan for Biomedical

Applications. J Nanomedic Biotherapeu Discover 2:102. doi: 10.4172/2155-
983X.1000102

Moist wound healing: a concept that changed our practice. (2004). Journal of Wound
Care., 13(6).

Montenegro, R., & Freier, T. (2010). Chitosan fiber

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Montenegro, Rivelino & R. G. Godeiro, José. (2012). Chitosan Based Suture – Focusing
On The Real Advantages Of An Outstanding Biomaterial.

Paul, W, & Sharma, C. (2004). Chitosan and Alginate Wound Dressings: A Short
Review. Trends Biomater. Artif. Organs, 18(1), 18-23.

Shah, A., & Goyal, R. (2008). Current Status of the Regulation for Medical Devices,
Indian Journal of Pharmaceutical Sciences, 70(6).

Singh, D., et al. (2000) Biomedical Applications of Chitin, Chitosan, and Their
Derivatives, Journal of Macromolecular Science, Part C: Polymer Reviews, 40:1, 69-
83, DOI: 10.1081/MC-100100579

Tachibana, M., et al. (1988). The Use of Chitin as a New Absorbable Suture Material -
An Experimental Study. Japanese Journal of Surgery, 18(5), 533-539.

Wan, A., et al. (2004). Mechanism of Fiber Formation by Interfacial Polyelectrolyte

Complexation. Macromolecules, 7019-7025.

Youndes, I., et al. (2015). Chitin and Chitosan Preparation from Marine Sources.
Structure, Properties and Applications. Mar Drugs. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4377977/.

43
 Glossary

 Biopolymer (noun)

o a polymeric substance (such as a protein or polysaccharide) formed in a

biological system

 Carapace (noun)

o a bony or chitinous case or shield covering the back or part of the back of

an animal (such as a turtle or crab)

 Catgut (noun)

o a tough cord made usually from sheep intestines

 Chitin (noun)

o a horny polysaccharide that forms part of the hard outer integument

especially of insects, arachnids, and crustaceans

 Chitosan (noun)

o a substance formed from chitin by partial deacetylation with alkali

 Crustacean (noun)

o any of a large class (Crustacea) of mostly aquatic mandibulate arthropods

that have a chitinous or calcareous and chitinous exoskeleton, a pair of

often much modified appendages on each segment, and two pairs of

antennae and that include the lobsters, shrimp, crabs, wood lice, water

fleas, and barnacles

 Interfacial Polyelectrolyte Complexation (noun)

44
 o a type of step-growth polymerization in which polymerization occurs at an

interface between an aqueous solution containing one monomer and an

organic solution containing a second monomer

 Litigate (intransitive verb)

o to carry on a legal contest by judicial process

 Polymer (noun)

o a chemical compound or mixture of compounds formed by polymerization

and consisting essentially of repeating structural units

 Shrimp (noun)

o any of numerous mostly small and marine decapod crustaceans (suborders

Dendrobranchiata and Pleocyemata) having a slender elongated body, a

compressed abdomen, and a long spiny rostrum and including some

(especially family Penaeidae) that are commercially important as food;

also : a small crustacean (such as an amphipod or a branchiopod)

resembling the true shrimp

 Suture (noun)

o a stitch made with a suture

o a strand or fiber used to sew parts of the living body

o the act or process of sewing with sutures

45
46
Carla Maridith D. Afuang
Address: 406 B. Francisco St., Barangka New Zaniga, Mandaluyong
City
Birthdate: May 25, 2000
Contact Number/s: 09205570609
Email: carlamaridith.afuang@gmail.com

EDUCATIONAL BACKGROUND

Secondary:
 City of Mandaluyong Science High School – E. Pantaleon St., Hulo, Mandaluyong
City
o June 2012 – (Present)

Primary:
 Good Shepherd Christian School – I. Lopez St., Mandaluyong City
o June 2006 – March 2012

ORGANIZATIONAL AFFILIATION/S
 Citizenship Advancement Training Officer (Academic Year 2015 – 2016)
 Science Gazette (Academic Year 2015 – 2016)
 MandSci Campus Integrity Crusaders (Academic Year 2015-2016)
 Biology Club (Academic Year 2017-2018)
 Fellowship Club (Academic Year 2017-2018)

SEMINARS ATTENDED
 3rd Biological Models Workshop
o University of the Philippines – Manila (January 2018)
 Job Hunting and Work Ethics
o Jose Rizal University (August 2017)
 Pre-Med Summit
o University of the Philippines – Diliman (March 2017)
 Integrity Development Workshop
o Office of the Ombudsman (Februray 2016)
 Ateneo Code: OD on Overdrive
o Ateneo De Manila University (January 2015)

CHARACTER REFERENCES

 Mr. Benjamin Ola

o City of Mandaluyong Science High School
 Mrs. Stephanie Fababair
o City of Mandaluyong Science High School

47
Jermaine B. Corpuz
Address: 534 M. Gonzaga Street, Mandaluyong City
Birthdate: September 13, 1999
Contact Number/s: 09218720135Email: maine.bca@gmail.com

EDUCATIONAL BACKGROUND

Secondary:
 City of Mandaluyong Science High School – E. Pantaleon St., Hulo,
Mandaluyong City
o June 2012 – (Present)

Primary:
 Good Shepherd Christian School – I. Lopez St., Mandaluyong City
o June 2006 – March 2012

ORGANIZATIONAL AFFILIATION/S
 Photography Club (Academic Year 2017-2018)
 Earth Science Club (Academic Year 2017-2018)

SEMINARS ATTENDED
 Job Hunting and Work Ethics
o Jose Rizal University (August 2017)

CHARACTER REFERENCES

 Mr. Edelyn Oben

o City of Mandaluyong Science High School
 Mr. Benjamin Ola
o City of Mandaluyong Science High School
 Mr. Paulo Salvidar
o City of Mandaluyong Science High School
 Mrs. Stephanie Fababair
o City of Mandaluyong Science High School

48
Patricia Beatrice S. Dela Cena
Address: 16 Int. 1, P. Oliveros st., Barangka Itaas, Mandaluyong City
Birthdate: December 04, 1999
Contact Number/s: 09283359897 / (02) 532-4902
Email: pb.delacena@yahoo.com

EDUCATIONAL BACKGROUND

Secondary:
 City of Mandaluyong Science High School – E. Pantaleon St., Hulo, Mandaluyong
City
o June 2012 – (Present)

Primary:
 St. Therese Private School – Sgt. Bumatay St., Mandaluyong City
o June 2005 – March 2012

ORGANIZATIONAL AFFILIATION/S
 Citizenship Advancement Training Officer (Academic Year 2015 – 2016)
 Science Gazette (Academic Year 2015 – 2016)
 Photography Club (Academic Year 2017-2018)
 Earth Science Club (Academic Year 2017-2018)

SEMINARS ATTENDED

 Job Hunting and Work Ethics

o Jose Rizal University (August 2017)
CHARACTER REFERENCES

 Mr. Benjamin Ola

o City of Mandaluyong Science High School
 Mr. Paulo Salvidar
o City of Mandaluyong Science High School
 Mrs. Stephanie Fababair
o City of Mandaluyong Science High School

49
Aubrey Lalaine A. Lomibao
Address: 170 Monday St., Mandaluyong City
Birthdate: September 20,2000
Contact Number/s: 09959311604
Email: alalomibao@gmail.com

EDUCATIONAL BACKGROUND

Secondary:
City of Mandaluyong Science High School – E. Pantaleon St., Hulo, Mandaluyong
City
June 2012 – (Present)

Primary:
 Dona Pilar C. Gonzaga Elementary School – M. Gonzaga St., Mandaluyong City
o June 2009 – March 2012
 San Felipe Neri Parochial School – Division of Mandaluyong
o June 2007 – March 2009
 Good Shepherd Christian School – I. Lopez St., Mandaluyong City
o June 2006 – March 2007

ORGANIZATIONAL AFFILIATION/S
 Citizenship Advancement Training Officer (Academic Year 2015 – 2016)
 Teknogham (Academic Year 2014 – 2017)
 Photography Club (Academic Year 2017-2018)
 FIlipino Club (Academic Year 2016-2018)

SEMINARS ATTENDED
 Job Hunting and Work Ethics
o Jose Rizal University (August 2017)
 Ateneo Fly High, Sci High!
o Ateneo De Manila University (July 2017)
 National Astronomy Week
o Rizal Technological University (February 2017)
 Engineering Symposium
o University of the Philippines – Diliman (January 2015)

CHARACTER REFERENCES

 Mr. Benjamin Ola

o City of Mandaluyong Science High School
 Mr. Paulo Salvidar
o City of Mandaluyong Science High School

50