Table of contents

Table of contents 摘　要 Abstract Symbols and Abbreviations: Preface I. Wounds II. Wound healing process III. Treatment and management of wounds IV. Collagen V. Collagen structure VI. Types of collagen VII. Properties of collagen VIII. Role of collagen in wound healing IX. Collagen peptides X. Sources of collagen XI. The Jellyfish Rhopilema esculentum XII. Aim and Objectives A. Aim (Broad objective) B. Specific Objectives XIII. Significance of the study XIV. Technical route CHAPTER ONE Extraction of Collagen from the Jellyfish Rhopilema esculentum. 1. Materials, chemical reagents and equipment. 1.1. Materials 1.2. Chemical reagents 1.3. Equipment 1.4. Solvent and buffer preparation 2. Experiment plan and method. 2.1. Experiment plan 2.2. Method 2.2.1. Collagen extraction 2.2.2. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 2.2.3. Fourier Transform Infrared (FTIR) Spectroscopy 3. Results. 3.1. Collagen extraction yield. 3.2. SDS PAGE Pattern of Collagen from the Jellyfish R. Esculentum 3.3. Fourier Transform Infrared (FTIR) Spectroscopy 4. Discussion. CHAPTER TWO Preparation of Collagen Peptides 1. Materials, chemical reagents and equipment. 1.1. Materials 1.2. Chemical reagents 1.3. Equipment 1.4. Buffer Preparation 2. Experiment plan and method. 2.1. Experiment plan 2.2. Method 2.2.1. Preparation of Collagen Peptides (CP1) 2.2.2. Preparation of Collagen Peptides (Cp2) 2.2.3. Tricine SDS-PAGE 3. Results 3.1. Collagen Peptides 3.2. Electrophoretic Pattern of Collagen Peptides 4. Discussion CHAPTER THREE In vitro study of Collagen Peptides 1. Materials, chemical reagents and equipment 1.1. Materials 1.2. Chemical reagents 1.3. Equipment 1.4. Medium and Buffer preparations 2. Experiment plan and method 2.1. Experiment plan 2.2. Method 2.3. Statistical analysis 3. Results and Discussion CHAPTER FOUR In vivo study of Collagen Peptides 1. Materials, chemical reagents and equipment 1.1. Materials 1.2. Chemical reagents 1.3. Equipment 2. Experimental plan and method 2.1. Experimental plan 2.2. Method 2.2.1. Experimental excision and wound creation 2.2.2. Animal grouping and treatment schedule 2.2.3. Immunohistochemistry 2.3. Statistical analysis 3. Results 4. Discussion Project summary Conclusion References Acknowledgements Published articles during the study period

Abstract

Background: Wounds represents a major health challenge as they consume a large amount of healthcare resources in order to improve patient’s quality of life. Many scientific studies are being conducted to find ideal clinical wound-healing biomaterials. Collagen have been proven to be a suitable candidate biomaterial. Marine as an alternative and safer source, has recently gained attention from many researchers. Several collagen-based biomaterials including collagen gel and collagen dressing derived from marine fishes, star fish, jellyfish, sponges etc. have been reported to have beneficial biological functions on wound healing. However, there is limited information regarding collagen peptides derived from jellyfish or the effects of these peptides on wound healing activity following oral administration. This study has investigated the potential wound healing effects of oral collagen peptides derived from the jellyfish. Methodology: In this study, collagen was extracted from the jellyfish Rhopilema esculentum using pepsin enzyme. SDS PAGE and FTIR was used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. In vitro wound scratch assay was done to determine the wound healing potential of the collagen peptides.  In vivo studies in mice were performed to determine the effects of collagen peptides in skin wound healing by examining wound closure, re-epithelialization and collagen deposition at skin wound sites. Confidence level (p<0.05) was considered significant using GraphPad prism software. Histopathological evaluation of fibroblast cells and collagen fibers of healed wound cuts was also performed. Results: The yield of collagen was 4.31%. The SDS-PAGE patterns and FTIR indicated that the collagen extracted from jellyfish was type I collagen.  Enzymatic hydrolysis of collagen I using collagenase II resulted to collagen peptides (CP₁) and alkaline proteinase/papain resulted to collagen peptides (CP₂). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25kDa. Wound scratch assay showed that there were significant effects on the scratch closure up on treatment with collagen peptides at a concentration of 6.25 µg/mL for 48hrs. Histopathological assessment results on peptides treated mice models showed remarkable fibrosis and collagenization. Conclusion:  Collagen peptides derived from the jellyfish Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future. So far, this is the first finding to be reported on the wound healing activity following oral intake of collagen peptides derived from the Jellyfish Rhopilema esculentum. Keywords: Collagen, collagen peptides, wound healing, jellyfish.

Symbols and Abbreviations:

APS	Ammonium persulfate
DMEM	Dulbecco’s Modified Eagle Medium
EDTA	Ethylene diamine tetra acetic acid
FBS	Fetal bovine serum
HE	Hematoxylin &eosin
HUVECs	Human umbilical vein endothelial cell
PBS	Phosphate buffered saline
SDS	Sodium dodecyl sulfate
PAGE	Polyacrylamide gel electrophoresis
TEMED	Tetramethylethylenedimine
FTIR	Fourier transform infrared spectroscopy
CP1	Collagen peptides sample 1
CP2	Collagen peptides sample 2
PDGF	Platelet derived growth factors
TGF β1	transforming growth factors β1
TGF β2	transforming growth factors β2
VEGF	Vascular endothelial growth factor
β-FGF	fibroblast growth factor β
ACE	Angiotensin converting enzyme
MMP	Matrix metalloproteinases
ECM	Extracellular matrix

Preface

The high risk of diseases associated with modern life style and increased life expectancy, are associated with increased incidence of major wounds that require medical intervention. Wounds are the type of injuries which happen relatively quickly in which skin is torn, cut or punctured. It may be caused by an act, such as falling, gunshot, or by infection following surgical procedures or by an underlying disease conditions. Wounds may be acute or chronic. Chronic wounds, such as venous and arterial ulcers, pressure ulcers and diabetic ulcers, are often associated with advanced age, compromised blood circulation, patient immobility, and systemic illnesses¹. According to the World Wound Care Markets 2008, chronic wounds (or skin ulcers) account for approximately 6 million skin wounds in the United States and 37 million skin wounds globally. Generally, chronic wounds are the challenge to health care system as they consume a great amount of healthcare resources. They are also a challenge to the wound care professionals who often adopt multidisciplinary approaches to achieve effective results. Conventional medicines including pain killers and antibiotics are the common measures for wound treatment. Some biomaterials such as collagen are known to be effective in protecting the wound from infection and maintain moisture in the wound area. Together with antibiotics they accelerate wound healing aiding shorter healing time, reduced hospital length of stay and costs and overall patient health improvement ². Collagen have long been used in wound care and management as a wound dressing material in various forms such as Powders, Amorphous gels/pastes, Gel-impregnated dressings, pads and rope type which is used for filling wound cavities³. Collagen is abundant in nature, it constitutes about 30% of animal protein. Over the years, bovine and porcine have been used as common source of collagen. However, the outbreak of bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE) and foot and mouth disease (FMD) that emerged from last decades have limited their use. Marine organisms such as fish, fish wastes, starfish, sponges and jellyfish have recently been utilized as the alternative source⁴. Collagen in nature is a large protein molecule which upon enzymatic hydrolysis produces very small bioactive peptides with interesting biological functions. Depending on their specific amino acid composition, bioactive peptides derived from collagen have been proven to show  immunomodulatory ^5,6, ACE Inhibitory properties^7–9, antibacterial ^10,11, antioxidative ¹² etcetra. Due to their high bioactivity and biocompatibility, collagen peptides may be useful in medicine and food industries. Although research on collagen peptides is growing rapidly, only few collagen peptide products have succeeded in the market such as Peptan^TM produed by Rousselot company ¹³ which is used for sports nutrition, nutricosmetics and cosmetic purposes. There is little or no information regarding jellyfish collagen peptides as a product used for wound healing properties in the market.

I.                   Wounds

Wounds occur when the skin is broken or damaged because of injury. Causes of injury may be the result of mechanical, chemical, electrical, thermal, or nuclear sources. The skin can be damaged in a variety of ways depending upon the mechanism of injury. Wound classification: Wounds can be classified according to the causes (open or closed) or according the healing time (acute or chronic). Regarding the underlying causes; open wounds (are the one in which blood escapes from the body and bleeding is clearly visible) are classified into:

• Superficial (on the surface) wounds and abrasions these are kind of wounds that leave the deeper skin layers intact. They are usually caused by friction rubbing against an abrasive surface.
• Cuts/ ulcerations this is the type of wound where all the layers of the skin and underlying tissues (muscle or bone) are cut.
• Punctured wounds are usually caused by a sharp pointed object entering the skin. E.g. stabbing a wound with a knife. Human and animal bites can be classified as puncture wounds, abrasions, or a combination of both.

For the closed wounds category, blood escapes the circulating system but remains in the body and these include; bruises, contusions and crush/compression injuries. Another kind of closed wound is pressure sores.  Pressure sores (bed sores) is the type of wound that can develop due to lack of blood supply to the skin caused by chronic pressure on an area of the skin. Diabetic patients, patients suffering from peripheral vascular diseases or malnutrition are at risk of developing pressure sores (Benjamin Wedro and Charles Patrick Davis, 2017 https://www.emedicinehealth.com/wound_care/page2_em.htm). Based on the physiology of wound healing, acute wounds are generally open wound and they usually complete the wound healing process within the expected time frame¹⁴. Chronic wounds are those result from a failure of normal progression through the wound healing process and therefore enter a state of chronic inflammation that either recurs frequently or requires a prolonged time to heal¹⁵.

II.                Wound healing process

Wound healing is an intricate biological process in which the skin, or any other tissue, repairs itself after injury. It is a complex and dynamic process that involves replacing devitalized and missing cellular structures and tissue layers on the wound. Normally, the process begins immediately after injury and can continue for months to years depending on the type of injury¹⁶. Wound healing involves four overlapping phases termed as hemostasis, inflammation, proliferation and remodeling, each with carefully regulated multiple sub steps that exhibit various interdependent relations. Important cells in wound healing process include:

• Platelets, which recruit inflammatory cells and form a provisional matrix.
• Macrophages, which regulate the cytokine environment in the wound, and influences proliferative responses and wound closure.
• Enzymes Matrix metalloproteinases (MMPs) particularly MMP-9 aids in phagocytosis, angiogenesis, cell migration during epidermal restoration, and tissue remodeling ¹⁵.

Following tissue injury, the initial response is usually bleeding. The cascade of coagulation and blood clotting stops the wound from bleeding leading to hemostasis (figure 1). This is accompanied by platelet aggregation which lead to the release of chemotactic factors from the platelets. Chemotactic factors including platelet derived growth factors (PDGF), transforming growth factors β₁ and β₂ (TGF β₁ & TGF β₂) which further recruit inflammatory cells to the wound site (inflammatory phase). Neutrophils leukocytes and macrophages are phagocytic in nature thus they clear the wound from foreign materials and bacteria that may infect the wound. Together with epidermal and dermal cells, inflammatory cells secrete other mediators that stimulate the chemotaxis of cells necessary for the proliferative phase. These mediators are mostly growth factors which regulate cell proliferation and stimulate cell migration within the wound bed. In response to hypoxic condition that was caused by the injury at the wound site, angiogenic factors including Vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and PDGF are produced and induce endothelial cells migration toward the wound site, where they proliferate, form cell-cell contacts and eventually form new blood vessels. Fibroblasts, keratinocytes, and smooth muscle cells also migrate and proliferate to re-epithelialize the denuded surface of the wound. Moreover, these cells synthesize and deposit a provisional extracellular matrix leading to wound contraction. During the final stage, the newly formed granulation tissue is remodeled by the activity of matrix metalloproteinases (MMP-9) balanced with tissue inhibitors of metalloproteinases (TIMPs), which rearranges the loose, regenerated dermis, and strengthens the repaired tissue^17,18.  Figure 1. Wound healing process

III.             Treatment and management of wounds

The purpose of wound care is to promote healing in the shortest time possible with minimal discomfort, pain, and scarring to the patient carried in a conducive environment for tissue regeneration and repair. Treatment strategy for chronic wounds focus mainly to correct the dysregulated physiological state of the wound and allow for healing to occur. Currrently, wounds are conventionally managed by using topical agents such as Povidone-Iodine, Mupirocin, and Silver-Sulphadiazine. Non-pharmacological approach to encourage wound healing processes are also applied. These include attending patient’s nutritional and hydration status and managing any co-morbidities that may have contributed to and/or caused ulcer development. Controlling  Infection by antibiotics, providing adequate wound oxygenation and wound debridement are also paramount approaches that are normally employed to maximize wound healing potential.¹⁴ Recently there are researches going on some growth factors which target the four phases of wound healing. Some of the growth factors are on clinical trials, for example pro angiogenic growth factor PDGF which accelerate wound healing by promoting angiogenesis. Also, enzymes which removes necrotic tissues on chronic wounds without causing blood loss are being tried for example NexoBrid (MediWound Ltd., Yavne, ISR) is a drug on phase III trial with both proteolytic activity and antibacterial activity making it an optimal agent for wound debridement. ¹⁴

IV.             Collagen

Collagen is a protein made up of amino-acids, which are in turn built of carbon, nitrogen, oxygen and hydrogen. It is the most abundant animal protein, accounting for almost 30% of total protein in animal body ¹⁹. It is the main structural material of the extracellular matrix of all connective tissues (i.e. skin, bones, ligaments, tendons and cartilage) as well as interstitial tissues of all parenchymal organs. Collagen is a very important protein in maintaining the biological and structural integrity of the extracellular matrix (ECM). It also provides the tissues with mechanical strength and physiological functions ^20,21

V.                 Collagen structure

Structurally, Collagen is composed of three polypeptide chains called α chains organized in a triple helical conformation. Each chain contains the repeating sequence of amino acids (i.e Gly-X-Y) _n, with Proline often located at positions X and Y. This special kind of arrangement  contributes greatly to the triple helical structure by restricting the dihedral angle of the main chain^22,23 . The three α chains can be identical or nonidentical. Identical α chains form homotrimers for example, collagen type II, contains three identical α chains [α1(II)]₃ and nonidentical α chains form hetero-trimers for example, collagen type I, contains two identical α chains and a third chain that differs, [α1(I)]₂α2(I). The α1 chain of one type of collagen (e.g. collagen I) has a primary structure different from that of the α1 chain of another type of collagen (e.g. collagen II). Collagens have non-triple helical domains at their N- and C-termini (Figure 2). These domains are called ‘non-collagenous’ (NC) domain ²². Naturally, the triple helix structure is stabilized by  high content of Glycine, Proline, Hydroxyproline, hydrogen bonding, and electrostatic interactions involving lysine and aspartate ²⁴. Collagen has the molecular weight of around 300,000Da, the diameter of about 14˜15 Å and it is 2800 Å long.  Figure 2. The structure of collagen

VI.             Types of collagen

There are 28 different types of collagen that are found in vertebrates (types I-XXIX) They can be broadly divided into two types; fibrillar and non-fibrillar collagen. The fibrillar collagen chains all have a perfect (Gly-X-Y) _n repeating sequence. In contrast, all non-fibrillar collagens have sites where the repeating tripeptide pattern is interrupted. Type I, II, III, V, and XI are fibril-forming collagens. Type IV collagen, is an example of non-fibrillar collagen. The different types of collagens are differently distributed in animal tissues and type I collagen accounts for about 80–85% of the total collagen in the body²⁵. The following are the common collagen types:

• Type I— skin, bone, dermis, tendon, ligaments, cornea
• Type II—cartilage, vitreous body, nucleus pulposus;
• Type III—skin, vessel wall, reticular fibers of most tissues (lungs, liver, spleen, etc.);
• Type IV—basement membranes,
• Type V—often co-distributes with Type I collagen, especially in the cornea

Fibril forming collagens (type I, II, Ill, and V) have large sections of homologous sequences independent of the organism, and constitute the most commonly used forms of collagen-based biomaterials for wound healing and tissue engineering purposes. ²⁶.  

VII.          Properties of collagen

Collagen has interesting physical and biological properties. Physical properties such as increased water absorption capacity makes it a good component for texturizing, thickening and gel formation. Moreover, collagen has the properties related to their surface behavior, which include emulsion, foam formation, stabilization, adhesion and cohesion, protective colloid function and film-forming capacity²⁷. Biologically, it has good biocompatibility, excellent degradability and weak immunogenicity. Purified collagen can be used as a wound dressing material and biomaterial for other medical purposes. Generally, collagen, collagen peptides and gelatin (denatured collagen) has been widely utilized in different fields including food industry, biomedical and Pharmaceutical industries, cosmetics, leather and film industries, diagnostic imaging and therapeutic delivery ²⁸.

VIII.       Role of collagen in wound healing

Collagen is an extremely important protein in wound healing due to its unique physical and biological properties that are essential for function. For instance, high tensile strength properties make it and important biomaterial in tissue repair. It has been used extensively in a wide range of medical fields, including wound healing, hemostasis, sutures, artificial heart valves and arteries, hernia repair, and soft tissue augmentation. Collagen type I is the most commonly used protein in wound healing biomaterial²⁹. Wound healing is a complex process involving cascade of events (phases) including platelet accumulation, inflammation, fibroblast proliferation, cell contraction, angiogenesis and re-epithelialization. This cascade ultimately leads to scar formation and wound remodeling. Collagen plays an important role in each of these phases of wound healing mainly due to its chemotactic role. In proliferation phase, it attracts cells such as fibroblasts and keratinocytes to the wound. This encourages debridement, angiogenesis and re-epithelialization. A chronic wound is stalled at one of these healing stages. This usually occurs during the inflammatory phase and is linked to elevated levels of inflammatory mediators and proteases such as matrix metalloproteinases particularly (MMP-8 and MMP-9) and elastases in the wound^30,31. These are the factors which in normal wound healing process, inflammatory mediators aid in cellular proliferation and migration and Protease enzymes play an important role in breaking down unhealthy Extracellular matrix (ECM) and induce the formation of new tissues. However, when these proteases are present in a wound at elevated levels for a prolonged period of time, they start to breakdown the healthy ECM leading to delayed wound healing, increase in wound size and sometimes the wound get infected ³. This is a common phenomenon in chronic wounds.Collagen use in wound dressing helps to shorten the healing time of chronic wounds by

• Decreasing the elevated protease activity of MMPs and elastases,
• Increasing fibroblast production at the wound site
• Maintaining the moisture, chemical and thermostatic microenvironment of the wound and minimize infection ³².

For many decades, collagen has been used in wound dressing due to these unique functions. currently, there are many collagen derived biomaterials that are employed in wound care and management.  Table 1 shows FDA approved list of some collagen-based products grouped into bovine, porcine and human origin and that are available in the market for wound care and management.   Table 1. Shows Collagen-based products used for wound care

Origin	Products	Company
	Catrix®	Lescarden Inc
	Cellerate®	Wound Care Innovations
	Collieva™	CollMed Laboratories
Bovine	Medifil®	BioCore
	Skintemp	BioCore
	Integra®	Integra Lifesciences
	Puracol	Medline Industries
	Colladerm	Innocoll
	Matriderm®	Suwelak
	ProHeal®	Suwelak
	Apligraf®
	Permacol®	Tissue Science Laboratroies
	Oasis®	Cook Biotech Inc.
	Biostep®	Smith and Nephew
Porcine	Colactive	Smith and Nephew
	Mediskin	Brennan Medical
	EZ-Derm™	Brennan Medical
	Biobrane®	Smith and Nephew
Equine	Septocoll®E	Biomet
	Decutastar®	ADL
Human	AlloDerm®	LifeCell Corp
	GraftJacket®	Wright Medical Technology
	Cymetra®	LifeCell Corp
	PromogranTM	Systagenix
Others	FibracolTM	Systagenix
	Promogran PrismaTM	Systagenix

IX.             Collagen peptides

These are small molecular weight peptides or protein molecules that are made by enzymatic hydrolysis of collagen. The amino acids composition of these peptides is similar to the parent collagen that is enzymatic hydrolysis disrupt only the bonds that held together the triple helix conformation, but the inner structure of collagen is preserved. Unlike collagen, Collagen peptides can easily be digested by gastric enzymes, absorbed and transported through PET-1 to systemic circulation where they can find their target and elicit their biological activities³³. In the skin, collagen peptides are known act as false collagen degradation peptides which sends a false signal in the fibroblast cells to synthesize new collagen fibers. Moreover, collagen peptides possess chemotactic properties they can promote cell migration and proliferation, an important process in wound healing³⁴.

X.                 Sources of collagen

Animals such as cows, pig and chicken are the common source of collagen but due to potential allergic and infectious risks arising from their use, scientists have found an alternative source for collagen with weak immunogenicity, good biocompatibility and biodegradable characteristics. One of the alternative source is the production of recombinant collagen using host models such as insects, yeast, mouse milk and plants ^35,36. In addition to safety, recombinant collagen preserves intact triple helix, an important structure for biological activity of which this activity is somewhat lost in the extraction process for animal derived collagen. Although recombinant technology produces high quality collagen, the whole process is more expensive and less affordable. Marine is another alternative source for collagen and bioactive substances that can be utilized in cosmetic, food, and pharmaceutical industry ³⁷. Several marine organisms have been used to extract collagen such as fishes ^38–40, star fish, jellyfish ^41–45, sponges ⁴⁶, sea urchin, octopus, squid, cuttlefish, sea anemone and prawn ⁴⁷. Most of the collagens extracted from marine animals have been characterized as collagen type I. Recently, it has  been reported that marine collagen I is a potential biomaterial for cell and tissue engineering ⁴⁸. Although research on marine collagen is rapidly growing, the application of this protein in medical field, pharmaceutical industry etc. is still in transition.

XI.             The Jellyfish Rhopilema esculentum

Certain edible large jellyfishes that belong to the order Rhizostomeae are consumed largely in China and Japan⁵. The jellyfish Rhopilema esculentum is the most common and high-yield aquatic animal in China. It is widely distributed over the South China Sea, the Yellow Sea and Bohai Sea and is abundant in the late summer to the early autumn. Due to its high nutrition values and pharmacological activities, it has been consumed by Chinese people for thousands of years for food and treatment of some diseases such as high blood pressure, bronchitis, tracheitis, gastric ulcers and asthma⁴². The jellyfish Rhopilema esculentum is very rich in proteins and minerals with negligible fat contents and low calories. Recently it has become popular in other countries due to its high collagenous protein⁴⁵. Zhuang et al⁹ reported that the peptides derived from R. esculentum could reduce the blood pressure in spontaneously hypertensive rats and be used as antihypertensive compounds in functional foods. Zhao⁴³ and Zhuang et al⁴⁹ reported that proteins isolated from jellyfish R. esculentum possess strong antioxidant activity and might be useful in the food and pharmaceutical industries. Cheng et al ⁴⁴ recently reported that collagen sponges derived from R. esculentum has potential hemostatic effects suggesting that it might be a suitable candidate for wound dressing applications. Other collagen-based biomaterials including collagen gel, films, and membranes have been reported to have beneficial biological functions on wound healing. However, the effect of collagen peptides on wound healing following oral administration is rarely concerned.

XII.          Aim and Objectives

A.    Aim (Broad objective)

The aim of this research is to investigate, the cutaneous wound healing activity of collagen peptides derived from the jellyfish Rhopilema esculentum.

B.     Specific Objectives

1. To extract collagen from Jelly fish Rhopilema esculentum by using Pepsin
2. To identify the extracted jellyfish collagen by FTIR method and SDS-PAGE.
3. To prepare jellyfish collagen peptides by enzymolysis method and their detection using Tricine-SDS PAGE.
4. To determine in vitro wound healing activity of jellyfish collagen peptides by using wound scratch assay.
5. To perform in vivo study by using excision and incision wound healing mice models.  
6. To determine histopathological changes following treatment with collagen peptides.

XIII.       Significance of the study

The study was conducted to investigate the wound healing potential of the collagen peptides derived from the jellyfish R. esculentum. The outcomes are expected to benefit China and the World at large by providing insight of the wound healing activities of the collagen peptides. It will also contribute in the growing scientific knowledge base about the beneficial wound healing effects of oral collagen peptides. Moreover, findings of this study will serve as reference for similar researches Worldwide. More importantly, this study may contribute towards drug discovery and health improvement by adding to the pool of available effective medications.

XIV.        Technical route

The figure below shows the technical summary of the entire project, from the start point to the end.  Figure 2. Technical route of the project entitled ‘THE WOUND HEALING POTENTIAL OF COLLAGEN PEPTIDES DERIVED FROM THE JELLYFISH Rhopilema esculentum’

CHAPTER ONE

Extraction of Collagen from the Jellyfish Rhopilema esculentum.

Collagen is insoluble in water. Its extraction from invertebrate tissues can only be possible by dissolving the raw material/tissue into weak acids or weak bases. Collagen structure is very strong stabilized by the hydrogen bonds and electrostatic interactions in the triple helix. Weak acids such as acetic acid is commonly used to extract collagen but with low yields. Moreover, collagen from fresh jellyfish is hard to extract because more than 95% in it is water. In order to increase extract yield, prior to enzyme treatment, the jellyfish tissue is first subjected to lyophilization to remove the water homogenization. Enzymes such as pepsin, papain, trypsin etc. are used to cleave strong bonds in collagen.  In this study, acetic acid was used as a solvent and pepsin enzyme was used to extract collagen from the jellyfish. SDS-PAGE and FTIR were used to identify the type of collagen and to determine the molecular weight.

1.      Materials, chemical reagents and equipment.

1.1.  Materials

Fresh filaments of jellyfish Rhopilema esculentum were bought from Auchan Supermarket in Nanjing City and transported in ice to the laboratory and stored at -80⁰C until use. Regenerated cellulose dialysis membrane (MWCO 50kDa) Sangon biotech

1.2.  Chemical reagents

Acetic acid	Nanjing chemical reagent Co. LTD
Pepsin (1:30000)	Dalian Meilun Biological technology co.Ltd
NaCl	Xilong scientific Co. LTD
Tris-base	Nanjing sunshine Bio
pH indicator paper	Shanghai SSS reagent Co.LTD
High molecular weight protein marker	TANON company
Sample loading buffer
Methanol	Jiangsu hanbon Sci & Tech. Co.Ltd
Sodium Dodecyl Sulfate (SDS)	Nanjing chemical reagent Co.Ltd
Ammonium persulfate (APS)
NaH2PO4.2H2O
Na2HPO4 .12H2O	Xilong scientific Co. LTD
HCl	Nanjing chemical reagent Co.Ltd
Glycine	Nanjing sunshine Bio

1.3.  Equipment

Homogenizer	JJ-2(2003-61)
Allegra 64R centrifuge	Beckman coulterTM
Freeze-dry machine (lyophilizer)	Telstar Lyoquest
pH meter	Sartorius co.ltd
5200 multi analysis instrument	TANON ShangHai. China
Stirrer

1.4.  Solvent and buffer preparation

0.1M Acetic acid	6ml Acetic acid diluted with distilled water to 1L
0.5M Acetic acid	30ml Acetic acid diluted with distilled water to 1L
0.05M Tris-base	6.06g Tris-base dissolved in 1L of distilled water
SDS-PAGE running buffer	3.02 g Tris-base, 18.8g Glycine, 1g SDS, 1L ddH2O
10% APS	0.1g Ammonium persulfate was weighed and dissolved into 1ml de-ionized water, labeled and stored at 40 C.
1M Tris, pH 8.8	12.1g Tris-base was weighed and dissolved into 100ml distilled water. HCl was added to adjust the pH to 8.8.
1M Tris, pH 6.8	12.1g Tris-base was weighed and dissolved into 100ml distilled water. HCl was added to adjust the pH to 6.8.
0.02 M sodium phosphate buffer (pH 7.2)	7 mg NaH2PO4.2H2O, 54.9 mg Na2HPO4 .12H2O dissolved into 10ml ddH2O
Distaining solution	500ml ddH2O, 400ml Methanol, 100ml Acetic acid

2.      Experiment plan and method.

2.1.  Experiment plan

The aim of this experiment was to extract collagen from Jelly fish Rhopilema esculentum, identify it.

2.2.  Method

2.2.1.Collagen extraction

The filaments of the jellyfish (203g) were rapidly rinsed with chilled tap water and then washed with pure water. The cleaned jellyfish filaments were then homogenized in 0.5 M acetic acid (2:1 v/w) for 2 min in an ice-water bath. Then 1% pepsin was added into this solution and the suspension was stirred for 3 days. The suspension was then centrifuged at 8000rpm for 15 min. The supernatant was salted-out by adding NaCl to obtain a final concentration of 2 M in the presence of 0.05 M Tris (at pH 7). The resulting precipitate was collected by centrifugation at 10000rpm for 20 min and then dissolved in 0.5 M acetic acid. The resulting solution was dialyzed against 0.1 M acetic acid and distilled water for 3 days. The dialysis solution was changed at least three times a day and the sample were lyophilized and stored at 4°C for further experimental use. The whole procedure was carried out at 4⁰C. The percentage yield of the extracted collagen was obtained by using the following formula: Yield of collagen wet%=weight of dry collagen gweight of wet jellyfish gX100 ……..⁵⁰

2.2.2.Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed according to the method of Barzideh et al ⁴¹ with slight modification. The gel consisting of an 8% resolving gel and 4% stacking gel was used. Collagen samples in freeze-dried form were dissolved in 0.02 M sodium phosphate buffer (pH 7.2) containing 1% SDS (w/v) and 3.5 M urea. They were then mixed with sample loading buffer at the sample buffer ratio of 1:1 (v/v) with or without DTT and heated for 5 min at 95 °C. A high molecular weight pre-stained marker was used to estimate the approximate molecular weight of collagen samples. Each sample (20 µg protein) was loaded into an 8% SDS-PAGE wells and run at 80 V for 30 min followed by 100 V for 2hr.

4% Stacking gel
ddH2O	2.7 ml
30% Polyacrylamide	0.67 ml
1M Tris, pH 6.8	0.5 ml
10% SDS	0.04 ml
10%APS	0.04 ml
TEMED	0.004 ml
8% Separating gel
ddH2O	3.3 ml
30% Polyacrylamide	2.7 ml
1M Tris, pH 8.8	3.8 ml
10% SDS	0.1 ml
10%APS	0.1 ml
TEMED	0.006 ml

Following electrophoresis, the gel was stained with Coomassie brilliant blue R-250 for two hours with constant slowly shaking on the shaker platform. The gel was then washed twice with tap water and the distaining solution was added. Distaining was changed every 1hr until the gel was clear with visible blue protein bands.  The gel was photographed by TANON 5200multi-gel image software, sample protein bands were compared with protein marker.

2.2.3.Fourier Transform Infrared (FTIR) Spectroscopy

The FTIR spectrum of Jellyfish collagen was recorded from 4000 to 650 cm^-1 at a resolution of 4 cm^-1 using FTIR spectrophotometer (Agilent Cary 640; Agilent Technologies, Santa Clara, CA, U.S.A.). Attenuated Total Reflexion (ATR) mode was used in this investigation. The FTIR spectrum obtained was analyzed using Resolutions Pro FTIR software (Agilent Technologies, Santa Clara, CA, U.S.A.)

3.      Results and Discussion.

3.1.  Collagen extraction yield.

The yield of Collagen extracted from jellyfish R. esculentumwas 4.31% (on a wet weight basis). This yield was higher than that reported for the same species (^42,44,49,51) using 1% pepsin. The appearance of lyophilized collagen was off-white spongy/mesh-like material as shown in figure 3.1 below.  Figure 3.1. Showing Collagen sponges extracted from the Jellyfish R. Esculentum

3.2.  SDS PAGE Pattern of Collagen from the Jellyfish R. Esculentum

SDS-PAGE was used to determine the molecular weight of the extracted collagen, type of collagen and the purity of the product. The gel showed three distinct protein bands at the molecular weight above 100kDa. From the literature, the molecular weight of a single collagen polypeptide chain is about 100kDa⁵². Thus, our electrophoretic pattern was similar to those found in other studies. SDS-PAGE showed that the extracted collagen consisted of two α chains between 100kDa and 150kDa, β (dimer) chain and γ (trimer) chain located on high molecular mass region (above 150 kDa) as shown in the figure 2. The α chains were the major component of Jellyfish collagen. α1 and α2 chains had different electrophoretic mobility, suggesting that their molecular weights were different. The SDS-PAGE pattern suggested that the Jellyfish collagen might be type I collagen which consisted of two α1 chains and one α2 chain as the major component. Moreover, Several studies have reported that marine invertebrate animals including Sea urchin (Paracentrotus lividus)⁵³ (Stichopus japonicus) starfish (Acanthaster planci)²⁰, squid (Uroteuthis duvauceli)⁵⁴ and jellyfish contain type I collagen. In addition, the electrophoretic pattern of our collagen sample was similar to those reported for collagen from the same species by Cheng et al ⁴⁴. The reducing conditions were tested to verify the presence of di-sulfide bonds in the protein chains which is among the characteristics of collagen type I. The gel showed that there is a significant difference between patterns of reduced and nonreduced samples, indicating that there is di-sulfide bond in the collagen. Amino acid analysis done by Cheng et al ⁴⁴ shows that collagen obtained from the Jellyfish R. esculentum contain cysteine, this confirms our SDS-PAGE results under reducing conditions of  DTT treated collagen samples. Under non-reducing condition, the pattern showed distinctive three polypeptide chains with less or no other protein band below 100kDa showing the purity of the sample.  Figure 3.2. Showing the electrophoretic patterns of Collagen extracted from the Jellyfish R. Esculentum. Lane 1: Molecular weight marker; Lane 2: Collagen sample without DTT; Lane 3: Collagen sample with DTT.

3.3.  Fourier Transform Infrared (FTIR) Spectroscopy

As shown in Figure 3.3, the FTIR spectra of the jellyfish Rhopilema esculentum collagen exhibited a characteristic peak of amide I, II, III and amide A, B. The amide A band position of collagen was found at ~3322.1 ^cm-1 and amide B at ~2926.8 ^cm-1 as a result of C-H stretching. The wavenumbers of the amide I, II and III bands are directly related to the configuration of collagen. The amide I band position of the Jellyfish collagen was observed at a wave number of ~1660.5 ^cm-1 which is, carbonyl group (C = O) stretching vibration coupled with COO- and Amide II peak was observed at ~1552.7 ^cm-1. Amide I and II peaks showed that jellyfish collagen had more molecular order. It could be suggested that pepsin disrupted a non-helical portion of telopeptide regions, leading to an increase in the molecular order of collagen structure. Amide III peak was observed at 1237.8 ^cm-1. Absorption peaks around 1400-1446 ^cm-1 were also found. These absorption peaks extensively corresponded to pyrrolidine ring vibration of proline and hydroxyproline. This result indicated that the helical structure of collagen was held together. Moreover, the FTIR spectrum of R. esculentum collagen, was found to be similar to FTIR spectra reported for the same species by Cheng X et al,⁴⁴. Altogether, the FTIR spectra suggest a well-maintained secondary structure in collagen extracted from R. esculentum.  Fig 3.3. Fourier Transform Infrared Spectrum of Collagen from the Jellyfish R.Esculentum

CHAPTER TWO

Preparation of Collagen Peptides

In this chapter we Used enzymes to break large polypeptide chains of collagen to small peptide chains. Tricine SDS PAGE was used to determine the molecular weight of these peptides.

1.      Materials, chemical reagents and equipment.

1.1.Materials

Type I collagen extracted from the jellyfish R. esculentum, dialysis membrane (MWCO 3.5kDa) Sangon biotech

1.2.Chemical reagents

Collagenase II	Worthington Biochemical corporation
Alkaline proteinase
Papain
NaOH	Xilong scientific Co. LTD
Tricine	Beijing solarbio Science & Technology Co., Ltd
Low molecular weight marker	Sigma–Aldrich Chemical Company
Sample loading buffer
Tris-base	Nanjing sunshine Bio
Sodium dodecyl sulfate (SDS)	Nanjing chemical reagent Co.Ltd
HCl	Nanjing chemical reagent Co.Ltd
Glycerol	Chinasun specialty products co.,LTD

1.3.Equipment

Water bath incubator		HH-S21-4-SShanghai cimo  co.LTD
5200 multi analysis instrument		TANON ShangHai. China
Allegra 64R centrifuge		Beckman coulterTM
Freeze-dry machine (lyophilizer)		Telstar Lyoquest
Microwave oven		Midea
Electronic balance (0.01mg)	Sartorius BT25S
pH meter	Sartorius co.ltd

1.4.Buffer Preparation

Sample loading buffer	1.25 ml of 1M Tris-HCl (pH 6.8), 0.5g SDS, 25mg bromophenol blue, 2.5ml Glycerol and DTT 0.077g/ml
Tricine buffer	1.45g Tris, 2.15g Tricine and 0.12g SDS dissolved in 120ml ddH2O
Tris buffer	24.22g Tris-base was weighed and dissolved into 1L distilled water. HCl was added to adjust the pH to 8.9
1M NaOH	400mg NaOH was weighed and dissolved into 10 ml distilled water.
6% acrylamide	0.6g Bis-acrylamide, 9.3g Acrylamide dissolved in 20 ml distilled water
3% Acrylamide	0.3g Bis-acrylamide, 9.6g Acrylamide dissolved in 20 ml distilled water
Gel buffer (3M Tris-HCl pH 8.45).	This was made by dissolving 36.4g Tris-base and 0.3g SDS in 100 ml distilled water. The pH was adjusted to 8.45 by adding HCl.
50% glycerol	l glycerol, 10 ml distilled water

2.      Experiment plan and method.

2.1.            Experiment plan

The aim of this experiment was to prepare collagen peptide from the collagen derived from Jellyfish Rhopilema esculentum.

2.2.            Method

2.2.1.      Preparation of Collagen Peptides (CP₁)

Samples (1g) of the extracted collagen were suspended in 200mL of distilled water and digested using collagenase II enzyme at an enzyme-to-substrate ratio of 1:20 (by mass). Digestion was carried out for 5 h at 37°C. The hydrolytic reaction was terminated by heating the samples at 95 °C for 10 min. The samples were then cooled to room temperature and centrifuged at3000 rpm for 30 min. The resulting supernatants were lyophilized as collagen peptides CP₁ and stored at 4°C for further experimental use.

2.2.2.      Preparation of Collagen Peptides (Cp₂)

Samples (1g) of the extracted collagen were suspended in 200 mL of distilled water (pH adjusted to 8.5 by adding 1M NaOH) at 55°C. 2.5% alkaline protease was added to the solution and the mixture was stirred for 2hrs, then the temperature was adjusted to 50°C and 3% papain was added to the mixture with stirring for 2hrs. The hydrolytic reaction was terminated by heating the samples at 95 °C for 10 min. The samples were then cooled to room temperature and centrifuged at5000 rpm for 15 min. The resulting supernatants were dialyzed against distilled water for 2hrs. The dialysis solution was changed every hour and the samples were lyophilized as collagen peptides CP₂ and stored at 4°C for further experimental use. The percentage yield of the collagen peptides product was obtained by using the following formula: Yield of collagen peptide %=weight of collagen peptides mgweight of collagen mgX100 …….⁵⁰  

2.2.3.      Tricine SDS-PAGE

The protocol for tricine-SDS PAGE was adapted from Nature protocols, 2006 with slight modification. The gel consisted of 6%resolving gel, 3% stacking gel and 3% preparative gel. Collagen peptide samples in freeze dried form were dissolved in distilled water and mixed with sample loading buffer at the sample buffer ratio of 1:1 (v/v) with Dithiotreitol and heated for 5 min at 95 °C. Each sample (2mg protein) was loaded into a well and run at 90 V for 2hr. A low molecular weight pre-stained marker was used to estimate the approximate molecular weight of collagen peptides.

Stacking gel
3% Polyacrylamide	0.3 ml
Gel buffer	0.93 ml
ddH2O	2.52 ml
10%APS	0.03 ml
TEMED	0.003 mL
Preparative gel
3% Polyacrylamide	0.32 ml
Gel buffer	0.5 ml
ddH2O	0.7 ml
10%APS	0.005 ml
TEMED	0.0005 ml (0.5µl)
Separating gel
6% Polyacrylamide	2 ml
Gel buffer	2 ml
50% Glycerol	1.3 ml
ddH2O	0.75 ml
10%APS	0.02 ml
TEMED	0.002 ml

Following electrophoresis, the gel was washed with tap water and incubated in a fixing solution for at least 20minutes with constant slowly shaking on the shaker platform. The gel was then washed twice with tap water and the staining solution was added. In order to activate the gel for staining the gel was heated for 10 secs in the microwave oven and then placed on the shaker platform for 2hr. The gel was then washed twice with tap water and the distaining solution was added. Distaining solution was changed every 1hr until the gel was clear with visible blue protein bands.  The gel was photographed by TANON 5200multi-gel image software, sample protein bands were compared with protein marker.

3.      Results

3.1.            Collagen Peptides

The digestion of jellyfish collagen using enzymes resulted into off white agglomerated powder of collagen peptides as shown in figure 3.1 below. On a dry weight basis, the percentage yield for CP₁ and CP₂ was 65% and 54% respectively.  Figure 3.1. Showing Collagen Peptides derived from the Jellyfish collagen

3.2.            Electrophoretic Pattern of Collagen Peptides

Fig shows the Tricine SDS PAGE of the hydrolyzed collagen. The electrophoretic pattern revealed the presence of collagen peptide chains with low molecular weight (Fig 3.2). CP₁ showed intensive collagen peptide bands at 10-15kDa while CP₂ showed the presence of collagen peptides with molecular weight <25kDa.  Figure 3.2. Electrophoretic patterns of collagen peptides: A) Lane 1:  Low Molecular weight marker; lane 2: Collagen peptides (CP₁). B) Lane 1:  Low Molecular weight marker; lane 2: Collagen peptides CP₂

4.      Discussion

The solubility of Collagen in water remains a challenge in all experiments it requires patience or otherwise only a little product yield will result. Different enzyme treatment on collagen resulted into different products. CP₁ sample has many small peptide chains with different molecular weights while CP₂ has few small peptides with molecular weight about 25kDa and 15kDa. We wondered if this disparity could influence their biological activity. Both products are from the same parent collagen, only enzyme digestion is different. Thus, we tested both products for invitro activity.

CHAPTER THREE

In vitro study of Collagen Peptides

The collagen peptides CP₁ & CP₂ were tested for their wound healing activity by using wound scratch assay. The principle of wound scratch assay follows culturing cells in a monolayer and when they reach about 80% confluency a group of cells is destroyed or displaced by scratching a line through the monolayer. The cells are then left to heal the artificial wound by themselves or with the aid of substances that can induce cell migration. For our case HUVEC cells were used to determine the potential of collagen peptides on cell migration.

1.          Materials, chemical reagents and equipment

1.1.            Materials

Cell line: Human embryonic vein endothelial cell line HUVEC was obtained from the laboratory at the engineering research center of peptide drug discovery and development, China Pharmaceutical University

CP1, CP2	Prepared in chapter two.
96-wells culture plate
0.22µl filters

1.2.            Chemical reagents

DMEM	GIBCO
Fetal bovine serum (FBS)	SiJiQing, HangZhou, China
Penicillin	Sangon
streptomycin	Sangon
Trypsin	Biosharp
NaCl	Xilong scientific Co. LTD
KCl,	Xilong scientific Co. LTD
Na2HPO4.12 H2O	Xilong scientific Co. LTD
KH2PO4

1.3.            Equipment

CO2cell incubator	Thermo scientific
Microscope	Shanghai Measurement Dimension Photoelectric Technology Co., LTD
Liquid Nitrogen container	West Chengdu Instrument Co., Ltd.
Electronic balance (0.01mg)	Sartorius BT25S
Life Countess cell counter	Life technologies
Autoclave	Shanghai three Medical Devices Co.,

1.4.            Medium and Buffer preparations

DMED medium	1 bag DMEM was first dissolved in 800 ml ddH2O, 50 mg penicillin, 100 mg streptomycin and 2 g sodium bicarbonate were added. ddH2O was then added to 1L. pH was adjusted to 7.4 and the solution was filtered and stored at 40C.
PBS solution	8 g NaCl, 0.2 g KCl, 3.63 g Na2HPO4.12 H2O, and 0.2 g KH2PO4 were dissolved in 1L ddH2O. the pH was adjusted to 7.4 by adding HCL. The solution was autoclaved and kept at 4˚C
0.25% Trypsin	0.25 g trypsin and 0.2 g EDTA were dissolved in 100 sterile PBS. The solution was filtered using sterile 0.22µl filters and stored in 4˚C

2.          Experiment plan and method

2.1.            Experiment plan

The aim of this experiment was to determine wound healing activity of collagen peptides derived from the jellyfish collagen by wound scratch assay.

2.2.            Method

HUVEC cells were cultured Immediately after taking them out from the liquid nitrogen. The cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS) supplemented with penicillin (120 U/ml) and streptomycin (75 mg/ml) at 37⁰C with 5% CO2. When the cells reached 80% confluency the cells were treated with trypsin for passing.

• After 3 passages, HUVEC cells were seeded in a 96-well plate and cultured as monolayer to confluence overnight. Each well contained approximately 1 X 10⁵ cells.
•  The monolayer was then scratched with a white 100 µl pipette tip to create an approximate 0.7 mm-wide wound area and washed twice with PBS to remove floating cells.
• After the line scratch, 100 µl DMEM was added into every well. To observe the effect of Collagen peptides on HUVEC cells migration, cells were incubated with collagen peptides (0-50 µg/ml) for various time periods (from 0 h to 48 h).
• Images of the wounded cell monolayer were taken using a microscope at 0, 18, 36 and 48h after scratched wounding.
• Cell migration activity was expressed as the percentage of the gap relative to the total area of the cell-free region immediately after the scratch, named the repair rate of scarification.

Sample preparation: 1 mg of each sample (CP₁, CP₂) was weighed and dissolved in 1 ml DMEM medium with 1%FBS. The solution was then filtered and diluted with 1%FBS-DMEM medium as follows (figure 2.2).  Figure 2.2. Serial dilution of the collagen peptides sample with DMEM medium Except for the first test tube, each test tube contained 350ul 1%FBS-DMEM. The highest intended concentration was 50µg/ml, and this was serially diluted. The following formula was used to find the initial volume to be diluted. C1V1=C2V2 C1=1mg/ml V1=X C2=50ug/ml V2=700ul Thus, x=C2V2C1 x=50ug/ml  X 700µl1mg/ml  , x= 35 µl   Calculation of Results To determine the surface area of the defined wound area the width (X) of the wounded area which was measured by the software in the microscope in mm and multiplied by the length which was 2.6 mm on a 200µm scale bar. Total Surface Area = X mm x length.        X   ̴0.7mm, length 2.6 mm. To determine the surface area of the migrated cells in to the wound area: -Migrated Cell Surface Area = length of cell migration (mm) x 2 x length Percent Closure (%) = Migrated Cell Surface Area / Total Surface Area x 100 Repair rate of scarification % = (the gap width of 0 hour – temporal gap width)/the gap width of 0 hour×100

2.3.            Statistical analysis

Two-way ANOVA was used to compare all groups to control groups. Data were presented as means ±standard deviation (SD) and statistical significant level was set at p values < 0.05. In the presented results significance levels are indicated as (*) for p <0.05; and (**) for p <0.01.

3.          Results and Discussion

Effects of collagen peptides on proliferation and migration of HUVEC cells are illustrated in Fig 3. The wound-healing scratch assay in which monolayers of cultured HUVEC cells were mechanically wounded with a white pipette tip indicated that collagen peptides promoted cell migration. The migration of HUVEC cells across the wound chasm was significantly enhanced in cells treated with collagen peptides compared to those treated with vehicle. The repair rate of scarification of collagen peptides was reached to 79.91% for CP₁ and 78.13% for CP₂ after 48 h compared to the vehicle treated cells which was 49.2%. For all concentrations 1.56-50µg/mL there was no obvious effect on the wound closure by sample treatment for 6hrs whereas significant wound closure effects of collagen peptides were shown after treatment for 18h, 36h and 48h compared with the control. The effect of collagen peptide on the wound closure was dose dependent where by the wound closure percentage for the cell treated with collagen peptides increased as the concentration increased from of 1.56µg/mL-6.25µg/ml (Fig 3A). the concentration more than 6.25µg/ml showed similar results to 6.25µg/ml. Fig 3B shows the repair rate of scarification at a concentration of 6.25µg/mL for 48hrs. The difference in rate of wound closure after 48hrs, at a concentration of 6.25µg/mL was statistically significant, for CP-1 against control (p<0.05) and CP-2 against control (p<0.05). Cell migration rate was based on the efficiency of monolayer cells invading the wound region with the collagen sample treatment for 0 to 48 h. Sample treatment significantly increased migration rate of cells into the wound area (Fig 3C.). As expected, the pre-wound region appeared narrower than the vehicle control, which had an obviously larger denuded area after 48h. After 48h the collagen peptides treated wound area was nearly closed while the control still had a wide gap. These results demonstrated that the collagen peptides from the jellyfish R.esculentum can induce cell migration. This might be due to the chemotactic effects of collagen peptides gave the cells ability to migrate to cover the artificial wound area. Moreover, the abundant amino acid residues in the collagen peptides could be a useful nutrient for cellular growth and proliferation and could provide a suitable environment to induce cell migration, although the real mechanism is still unclear. Figure 3. Showing the In Vitro wound healing activity of Collagen Peptides derived from the Jellyfish collagen.   

CHAPTER FOUR

In vivo study of Collagen Peptides

Although our samples showed a good wound healing activity on HUVEC, we wanted to study in vivo activity of our samples. In vivo study is a very important stage of drug discovery/development. At this stage there are a lot of factors that might affect bioavailability and even bioactivity of the drug. Some small molecular weight collagen peptides are resistant to GIT enzyme digestion and metabolism thus they can easily be absorbed with less alterations and transported to their target cells (fibroblast) where they elicit their wound healing activity. In this study, mice models were used for wound healing activity and we chose intragastric as the route of administration for our samples. Animals: Adult male mice with body weight of approximately 26g each were used in this study. The mice were maintained under standard acclimatization conditions of 12h light/dark circle at approximately 25⁰ C temperature and were provided with standard rodent food. All the experimental protocols to use animals were approved by the animal care and use committee at China Pharmaceutical University.

1.      Materials, chemical reagents and equipment

1.1.Materials

CP1, CP2	Prepared in chapter two
Rodent food
Normal saline (0.9% NaCl)

1.2.Chemical reagents

pentobarbital sodium
Hydrogen peroxide
Paraffin
Sodium citrate buffer
Anti-basic fibroblast growth factor (bFGF) antibody
Antirabbit IgG
Methanol
Formalin

1.3.Equipment

Balance scale
Microscope
Vernier caliper
Shaving machine
Scissor, forceps

2.      Experimental plan and method

2.1.Experimental plan

The aim of this experiment was to determine the in vivo wound healing activity of jellyfish collagen peptides by using excision and incision wound healing mice models and assess the activity by immunohistochemistry and histopathologicalobservations.

2.2.Method

2.2.1.      Experimental excision and wound creation

The mice were anesthetized with intraperitoneal injection containing 1% pentobarbital sodium (0.25 ml/26 g body weight). The dorsal hair of the mouse was removed by a shaving machine to prepare the back skin for generation of a standardized full-thickness cutaneous wound. An excision wound of approximately 10mm diameter was made by removing a full-thickness piece of skin using a scissor from a pre-determined shaved area. The wounds were left undressed to the open environment without any local or systemic anti-microbial agents administration. The cages were placed in a cool environment at room temperature ̴25⁰C until mice fully recovered from anesthesia which was the next day (post wound day).

2.2.2.      Animal grouping and treatment schedule

Wounded mice were randomly divided into 7 groups of 10 mice each, that is the control group, CP₁ and CP₂. The collagen peptides (0.3, 0.6 and 0.9 g kg^_1 body weight) or normal saline as a vehicle were administered orally (intragastric) every morning for 6 days. The wound contraction size was observed and measured on each day of treatment. Photos were taken using digital camera at day 1 and 8. At day 6 and 8 three mice with larger wound area were sacrificed from group and their healing skins were collected and put in the formalin solution for further analysis.

2.2.3.      Immunohistochemistry

Paraffin-embedded tissues were sectioned (5 mm), and antigen retrieval was performed using 10 mM sodium citrate buffer. Endogenous peroxidase activity was blocked by treating sections with 0.3% hydrogen peroxide in methanol for 24hr. Tissues were treated with anti-basic fibroblast growth factor (bFGF), antibody overnight at 48C. Specific labelling was detected with a peroxidase-conjugated goat antirabbit IgG and avidin-biotin peroxidase complex. Slides were then mounted with coverslips and analyzed by two blinded pathologists. bFGF was assessed by immune-reactive cell density plus expression intensity. Immuno-reactive cell density was graded semi-quantitatively as 1) less than 10% per field, 2) 10_30% per field, 3) 30_70% per field, or 4) more than 70% per field, whereas expression intensity was graded as 1) mild, 2) moderate, or 3) severe.

2.3.Statistical analysis

Two-way ANOVA was used to compare all groups to control groups. Data were presented as means +SEM and analyzed with Prism software (GraphPad prism version 5) and statistical significant level was set at p values < 0.05. In the presented results significance levels are indicated as (*) for p <0.05; and (**) for p <0.01.

3.      Results

General observations. All mice appeared healthy throughout the study except for the day they were wounded most of the mice had minor weight loss due to pain caused by the wound. From the second day, when the treatment started all the mice regained their weight and the wound area started to decrease. From the graph (figure 3B)., There was no significant difference observed on the mice body weight indicating that our sample did not have negative effects on mice appetite and health in general. The significant effects of wound healing on collagen peptides treated mice were shown after 5 days of treatment for CP₁ while CP₂ treated mice had similar results to the control (Figure 3A). On day 6, CP₁ treated mice had increased wound contraction compared to control (P value <0.001) implying that collagen peptides could improve the wound healing in a shorter time. From our observations, the wound healing time was 5-7 days, at this time, most mice in CP₁ group had smaller wound area which was almost closed compared to the other groups (Figure 3C, D). Histological analysis of the wounded skin showed increased signs of re-epithelialization and new tissue formation in collagen peptides treated groups compared to the vehicle group. Immunohistochemistry showed increased expression of β-FGF and TGF-β in collagen peptides treated mice than the vehicle group.  Figure 3C. showing images of mice groups with full thickness excised wounds at day 0.      Figure 3D. Showing images of contracted wound area on mice following intragastric administration of collagen peptides or vehicle 6 days after treatment.               

4.      Discussion

The aim of this study was to investigate the wound healing potential of jellyfish collagen peptides. Wound healing is a complex process with four distinct phases working interdependently. Since collagen is known to interact with all phases, in this study we examined different roles the collagen played part or contributed to the wound healing by examining the mice skin section observing collagen deposition, tissue regeneration and re epithelialization, β-FGF and TGF-β expression. Collagen deposition was noticed to be more in collagen peptides treated mice (both CP₁ and CP₂ at a dose of 0.9g/kgbw) than in vehicle treated group. this can be marked from Masson trichome skin section where collagen fibers appear blue and on H&E skin section the collagen fibers appear pink or light pink marking fibrosis. Fibrosis is the process that results into formation of excess tissue fibers on the skin especially following injury, marking wound healing. Masson and H&E skin sections also showed sign of tissue regeneration. This a process of repairing or forming new tissues that replace the wounded tissue structures. From the skin sections the regenerated tissues can be easily observed, they are basophilic in shape with nucleus located at the center (arrow). These features were marked more in collagen treated mice than in vehicle group indicating that collagen peptides enhanced tissue regeneration process. moreover, treatment of mice with collagen peptides resulted into increased re-epithelialization. This is the process of formation of epithelial tissue that covers the wound and allow it to heal. From the skin sections this process can be marked by increased expressions of TGF-β and β-FGF. TGF-β is one of the chemotactic factor that play role in inflammatory phase by recruiting more inflammatory cell to the wound site. β-FGF stimulate fibroblast proliferation and promote these cells to re-epithelize the altered surface of the wound. Proliferating cells in turn, synthesize and deposit a provisional extracellular matrix leading to wound contraction. Moreover β-FGF stimulate formation of new blood vessels to increase nutrient and oxygen supply to the wound^17,18. The expression of these two factors were observed on the skin section and marked with cytoplasmic light brown bodies as a resulting reaction with their specific antibodies (arrow). Putting all together, we can conclude that oral administration of collagen peptides enhanced wound healing.

Project summary

All the necessary experiments were carried out successfully and results were positive according to the objectives of this project. The Collagen extracted from the jellyfish R. esculentumwas type I collagen which was off-white in color and spongy/mesh-like in appearance. The digestion of jellyfish collagen using enzymes resulted into off white agglomerated powder of collagen peptides CP₁ and CP₂ with molecular weight 15kDa and 25kDa respectively. As expected, collagen peptides were effective in inducing cellular migration an important process in wound healing which was observed in HUVEC cells with a minimum concentration of 6.25ug/ml for 48 hrs. Oral administration of these collagen peptides (CP₁ and CP₂) at a dose of 0.9gkg ^-1 to the mice bearing full thickness excised wounds resulted into increased wound contraction as compared to the control group.

Conclusion

Collagen is an interesting protein which can be broken down into small different proteins with various biological activity. The jellyfish Rhopilema esculentum is one of the marine organisms that can serve as safe collagen source. In addition to reported nutrition benefits and other pharmacological values, collagen peptides derived from the jellyfish Rhopilema esculentum have potential to accelerate wound healing thus could be a promising product for wound treatment in the future.

References

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