FLUORINATED METHACRYLAMIDE CHITOSAN HYDROGEL IMPROVES
CELLULAR WOUND HEALING PROCESSES
A Thesis Presented to The Graduate Faculty of The University of Akron
In Partial Fulfillment of the Requirements for the Degree Master of Science
Sridhar Akula December, 2016 i FLUORINATED METHACRYLAMIDE CHITOSAN HYDROGEL IMPROVES
CELLULAR WOUND HEALING PROCESSES
Sridhar Akula
Thesis
Approved: Accepted:
Advisor Interim Dean of the College Dr. Nic D Leipzig Dr. Donald P. Visco Jr.
Committee Member Dean of the Graduate School Dr. Lingyun Liu Dr. Chand K. Midha
______Committee Member Date Dr. Jie Zheng
Department Chair Dr. H. Michael Cheung
ii ABSTRACT
Wound healing is a complex process with many local and systematic factors
affecting it, including advancing age, local tissue oxygen tension, stress, diabetes etc. Some
of these factors severely decelerates wound healing processes and lead to non-healing
chronic wounds. In extreme cases affected areas are amputated such as in the case of
diabetic foot ulcers. A key underlying problem in chronic wounds is the low availability of oxygen, which leads to stalled wound healing. Current clinical therapies to treat non- healing chronic wounds are hyperbaric oxygen therapy and topical oxygen therapy, but
they are not economically viable and inconvenient for the patient. Simple solutions are
required to treat chronic wounds in an economically viable way to overcome the
shortcomings of these currently available clinical therapies. Here, I propose a chitosan- based hydrogel incorporating perfluorocarbons (PFCs) that has been named fluorinated methacrylamide chitosan (MACF), which can supply oxygen to chronic wounds. Previous work from the Leipzig lab has demonstrated that MACF hydrogels can be loaded with oxygen and can supply it locally to oxygen-deficient environments.
This thesis presents in vitro studies testing the potential benefits of oxygen releasing
MACF hydrogels on human skin cells (human dermal fibroblasts and human epidermal keratinocytes), evaluated under both normoxic (21% O2) and hypoxic (1% O2)
environments. Results showed that MACF improved cellular functions involved in wound
iii healing such as cell viability (metabolism), cell migration and total cell number under
hypoxic conditions in both human dermal fibroblasts and human epidermal keratinocytes.
Adenosine triphosphate (ATP) quantification also revealed that MACF treatments improved cellular ATP levels significantly over controls under both normoxia and hypoxia.
These studies indicate that supplying local oxygen via MACF under hypoxic environments improves cellular functions involved in wound healing processes and MACF will be a promising solution in improving chronic wound healing.
iv ACKNOWLEDGEMENTS
Firstly, I would like to thank Dr. Nic D Leipzig for his guidance, support and encouragement throughout my research. I am extremely grateful for the opportunity to be part of your laboratory. I would like to thank my committee members: Dr. Jie Zheng, and
Dr. Lingyun Liu for their assistance, valuable suggestions and time. They have also been my trusted advisors with my career and personal ambitions. I value their support and counsel and hope to be able to continue our relationships. I am also grateful to National
Institutes of Health for their financial support (Grant R15GM10485).
I would like to thank the University of Akron’s Chemical and Biomolecular Engineering department and the Department of Polymer Science and other laboratory/research groups for granting access to their laboratory equipment. I also like to thank the members of our laboratory/research group both past and present (Pritam Patil, Mahmoud Farrag, Trevor
Ham, Andrew McClain, Ashley Wilkinson and Hang Li) who have helped me through the years. I wish you all the best in your endeavors. Lastly, I would like to thank my parents,
Akula Kumar and Vijayalakshmi, for their love, support, and encouragement through my growth and never losing faith in me, thank you for being my pillars of support. Moreover,
I truly thank my siblings and friends, for their encouragement and help on my research.
v TABLE OF CONTENTS
Page
LIST OF FIGURES……………………………………………………………… ix
CHAPTER
I SPECIFIC AIMS………………………….………………. 1
II BACKGROUND………………………….………………. 3
2.1 Wound healing……………….….………………………… 4
2.2 Factors that affect wound healing and Introduction to chronic wounds………...... 5
2.3 Importance of oxygen in wound healing………………………………………………...... 6
2.4 Biomaterials for dermal wound healing………...... 7
2.5 Current Therapies to Treat Chronic wounds………...... 9
2.6 Artificial oxygen carriers and oxygen generating biomaterials………………………………………………... 11
2.7 Chitosan and its properties…………………...... 13
2.8 Hydrogels………………………………………………….. 14
2.9 Fluorinated Methyl Acrylamide Chitosan (MACF)…….… 15
2.10 Introduction to in vitro wound healing assays…………. 17
III EXPERIMENTAL DESIGN………………………….... 21
3.1 Preparation of fluorinated methacrylamide chitosan and methacrylamide chitosan polymers……………………….. 21
vi 3.2 Hydrogel Preparation……………………………………… 22
3.3 Saturating MACF hydrogels with oxygen………………… 23
3.4 Effect of Oxygenated MACF gel on cell media…………… 24
3.5 Cell culture………………………………………………… 24
3.6 Cell proliferation studies………………………………….. 25
3.6.1 Presto blue to evaluate cell metabolism (viability)………. 26
3.6.2 Digesting the cells in lysozyme………………………….. 26
3.6.3 Micro BCA assay to evaluate protein synthesis…………… 27
3.6.4 PicoGreen assay to determine total double stranded DNA... 27
3.6.5 Adenosine triphosphate (ATP) quantification…………….. 28
3.7 Scratch assay to study cell migration……………………… 29
3.8 Immunohistochemistry procedures………………………... 30
IV RESULTS…………………………………………………. 31
4.1 Effect of oxygenated MACF gel on cell media……………. 32
4.2 2 Effect of oxygenated MACF gels on cell morphology, metabolism and protein synthesis…………………………. 33
4.3 Effect of oxygenated MACF on total double stranded DNA and ATP levels……………………………………………. 37
4.4 Effect of oxygenated MACF on cell migration…………… 38
4.5 Ki67 IHC staining (Protein embedded with cellular proliferation) ……………………………………………… 41
V DISCUSSION……………………………………………. 43
VI CONCLUSIONS and FUTURE WORK…………………. 48
REFERENCES…………………………………………………………………... 50
vii APPENDIX………………………………………………...... 57
viii LIST OF FIGURES
Page
2.1 A.Phases of wound healing; B. Figure representing overlapping of wound healing phases……………………………………………………………… 5
2.2 A. Hyperbaric Oxygen therapy; B. Negative pressure wound therapy / topical oxygen wound therapy…………………………………………….. 9
2.3 Pentadecafluorooctanoyl chloride (Example of PFC)……………………… 11
2.4 Structure of chitosan……………………………………………………….. 13
2.5 A. Sample hydrogel B. Effect of water on hydrogel…………………….. 14
2.6 Example MACF (Chitosan modified with Pentadecafluorooctanoyl chloride (PFC)) ……………………………………………………………. 15 3.1 A. Reactions involved in the preparation of MACF. Step1: -Addition of PFCs to chitosan. Step2: Methacrylation. B. Figure representing oxygen molecules loaded to the MACF hydrogels by Van der Waal’s forces and reloading it at the necessary places such environments……………………’ 22
3.2 Schematic diagram representing steps involved in scratch assay………….. 29
4.1 High resolution 19F spectra for MACF and B. High resolution 1H spectra for MACF………………………………………………………………….. 31
4.2 Oxygenated MACF gels elevate PO2 level in culture media. PO2 level is measured in the media with oxygenated MACF gel as compared with MAC and no gel media at time several time points up to 48 h. Mean+ SD with n=4………………………………………………………………………… 32
ix 4.3 Oxygenated MACF gels elevate PO2 level in culture media. PO2 level is measured in the media with oxygentaed MACF gel as compared with MAC and no gel media at time several time points up to 48 h. Mean+ SD with n=4Effect of MACF on human dermal fibroblast morphology. Representative phase contrast images of human dermal fibroblasts grown under different treatments for 72 h. A,B,C are in normoxia (21% O2) and D,E,F are in hypoxia (1% O2). Scale bar is 200 µm………………………… 33
4.4 Effect of MACF on human epidermal keratinocytes morphology. Representative phase contrast images of human dermal fibroblasts grown under different treatments for 72 h. A, B, C are in normoxia (21% O2) and D, E, F are in hypoxia (1% O2). Scale bar is 200 µm…………………………………………………………………………. 34
4.5 The effect of MACF on cell metabolism, as determined by the PrestoBlue assay. MACF improved cell metabolism (viability) under both hypoxic and normoxic environment, as showed by increased reducing power per well (equivalent DTT concentration). A. Human dermal fibroblasts and B. Human epidermal keratinocytes. White bars represent normoxic environment and black bars represent hypoxic environment. Significant difference at p < 0.0001. Letters are significantly different from one another by single-factor ANOVA (p < 0.0001). All data n = 3-4, mean ± SD……… 35
4.6 The effects of MACF on total DNA and total ATP levels. MACF increased total DNA level and total ATP level per well under both hypoxic and normoxic environments. A, C are human dermal fibroblasts and B, D human epidermal keratinocytes. A, B represent total DNA levels measure by PicoGreen assay and C, D represent total ATP level measured via luminescent assay. White bars represent normoxic environment and black bars represent hypoxic environment. Letters are significantly different from one another by single-factor ANOVA (p A, C < 0.005; B, D p < 0.0001. All data n = 3-4, mean ± SD……………………………………………..… 37
4.7 Oxygenated MACF accelerated cell migration under hypoxia in both human dermal fibroblasts and human epidermal keratinocytes. A. Human dermal fibroblasts B. Human epidermal keratinocytes. White bars represent normoxic environment and black bars represent hypoxic environment. Letters are significantly different from one another by one-factor ANOVA (p < 0.0001). All data n = 3-4, means ± SD……………………………….. 38
4.8 Effect of MACF on human dermal fibroblast migration. Representative phase contrast images of scratch assay A. Scratch at t=0 for all controls; B-G images of scratch at t= 24 h; B, C, D are in normoxia (21% O2) and E, F, G are in hypoxia (1% O2). Scratch is completely closed in normoxic controls by 24 h whereas
x as in hypoxic controls it is not completely closed with MACF showing 39 improved human dermal fibroblast migration. Scale bar is 200 µm………
4.9 Effect of MACF on Human epidermal keratinocytes migration. Cell migration is studied by in vitro scratch assay. A. Scratch at T=0 for all controls; B-G images of scratch at t= 24 h; B, C, D are in normoxia (21% O2) and E, F, G are in hypoxia (1% O2). Scratch is completely closed in normoxic controls by 24 h whereas as in hypoxic controls it is not completely closed with MACF showing improved human epidermal keratinocytes migration. Scale bar is 200 µm……………………………. 40
4.10 IHC staining of ki67 protein which is a cell marker for cell proliferation on human dermal fibroblasts. Representative fluorescent images; A, C are images of scratch under Normoxia whereas B, D are under Hypoxia. A, B are images taken on the scratch whereas C, D are taken outside the scratch. Scale bar is 200 microns……………………………………………………. 41
4.11 IHC staining of ki67 protein which is a cell marker for cell proliferation on human epidermal keratinocytes. Representative fluorescent images; A, C are images of scratch under Normoxia whereas B, D are under Hypoxia. A, B are images taken on the scratch whereas C, D are taken outside the scratch. Scale bar is 200 microns……………………………………….…. 41
xi CHAPTER I
SPECIFIC AIMS
Besides being important for normal metabolism, oxygen plays an important role in
wound healing processes[9]. In normal dermal tissues PO2 levels are 25-40 mmHg, but in in some chronic wounds it may decrease to as low as 5 mmHg of PO2 and has been
correlated with decrease in dermal wound healing process/responses[10, 11]. To overcome
this deficit and resulting negative effects, the Leipzig lab has have developed a potential
simple solution called MACF (fluorinated methacrylamide chitosan). Oxygenated MACF
gels can supply oxygen to the places where it is needed like a chronic wound[7]. I am
planning to study the effect of our MACF gels on in vitro human dermal fibroblasts and
epidermal keratinocytes to reverse negative effects of hypoxia as compared to normoxic
controls.
AIM 1: To study the effect of oxygenated MACF hydrogels on human dermal fibroblast
and keratinocyte cell migration
This Aim will employ scratch wound assay model where a scratch is made on a
confluent cell monolayer. For this Aim, I hypothesize that MACF hydrogels will provide
additional oxygen to improve/enhances the cell migration in cells that are grown in hypoxic
environments. It is well-known that hypoxia decreases cell migration responses, thus
increasing the time required for cells to migrate and close a wound. In this Aim I will study
1 the effect of our oxygenated MACF gels on cell migration of human dermal fibroblasts, and human epidermal keratinocytes in a hypoxic environment (1% O2) as compared to normoxic responses.
AIM 2: To study the effect of oxygenated MACF hydrogels on human dermal fibroblast and keratinocytes cell proliferation, cell metabolism, protein synthesis and Adenosine tri phosphate (ATP) production
Both cell types will be seeded in 24 well plates and allowed to proliferate in both hypoxia and normoxia and treated with or without oxygenated MACF hydrogels for 3 days.
After 3 d, assays will be performed for cell proliferation, cell viability, cell quantification, protein synthesis, cell metabolism and ATP content using commercial kits. Here I hypothesize that supplying oxygen using our MACF hydrogels will improve all cell functions in human fibroblasts and keratinocytes grown in both hypoxia and normoxia.
Cells in hypoxia will readily respond to the MACF treatments. It is established that low levels of oxygen in dermal chronic wounds is the reason for poor wound healing responses[9].
In all of my studies I will be using six different treatments to compare the overall effect of
MACF gels to no gel treatment and MAC treatment in both normoxia and hypoxia environments. Additionally, in all in vitro studies two levels of cell type will be used: human dermal fibroblasts and human epidermal keratinocytes.
2
CHAPTER II
BACKGROUND
Wounds are typically classified as chronic wounds when they require more than 6
weeks to reepithelize (close). Chronic wounds are extremely problematic to affected
patients in terms of both financial as well as quality of life. The following statements
quantify the impact of chronic wounds in USA, “in the United States, chronic wounds
affect around 6.5 million patients. It is claimed that an excess of US$25 billion is spent
annually on treatment of chronic wounds and the burden is growing rapidly due to
increasing health care costs, an aging population and a sharp rise in the incidence of
diabetes and obesity worldwide. The annual wound care products market is projected to
reach $15.3 billion by 2010”[12]. Additionally, “more than 60% of nontraumatic lower- limb amputations occur in people with diabetes. In 2006, about 65,700 non-traumatic lower-limb amputations were performed in people with diabetes”[13]. These two
statements highlight the need for chronic wound treatments. One main problem underlying
many chronic wounds is low tissue oxygenation levels, which leads to a self-sustaining inflammatory phase during wound healing, preventing the progression of wound healing.
This eventually leads to non-healing chronic wounds. This phenomenon of low oxygen
availability is expected as most of the cells store biochemical energy in the form of ATP
and this ATP is produced through oxidative phosphorylation[9, 14]. Existing therapies
such as hyperbaric oxygen therapy and topical oxygen therapy are either expensive or
3
inconsistent in treatment, poor patient compliance as well as some toxicity issues[9, 15,
16]. PFCs incorporated chitosan polymer hydrogel, which can be loaded with oxygen and supply it to necessary places such as chronic wounds, could provide a promising solution
to accelerate wound healing in non-healing chronic wounds[7, 17].
2.1 Wound healing
Wound healing is an intricate process where the skin (or another organ-tissue) repairs itself after an injury. It occurs in four phases: hemostasis (blood clotting), inflammation, proliferation and remodeling (maturation) as shown in figure 2A. All these
phases are overlapping, and various locations within a wound may be at different phases
of wound healing (figure 2B)[9, 14, 18]. The hemostasis phase starts within minutes of the
occurrence of the wound, in this phase platelets adhere to the wound and release chemical
signals that help in releasing fibrin, which acts as glue and binds platelets, thus helping
blood to clot and stop the bleeding[9]. The hemostasis phase is followed by the
inflammation phase where phagocytosis occurs. During phagocytosis white blood cells
clear the damaged and dead cells along with bacteria, pathogens and debris in the wound.
Another important aspect in this phase is the release of platelet derived growth factors that
help in clearing the path for new cells by proliferation and migration in the proliferative
phase[9]. In the proliferative phase, angiogenesis (formation of new blood vessels),
collagen deposition, granulation tissue formation, epithelialization, and wound contraction
occur, thus helping in contracting the wound surface area[9, 14]. Finally, the wound is
healed completely during the remodeling phase, which is the final phase which may last
from 6 months to 2 years depending on the size and location of the wound[9]. In remodeling
4
phase mainly provisional collagen (type 3) is replaced with more stable type 1 collagen
which increases wound tensile strength[9]. All these phases play a vital role in wound
healing process and disruption occurring in any of the cellular process in these phases leads
A B
Figure 2-1. A. Phases of wound healing[1] ; B. Figure representing overlapping of wound healing phases[5].
to non-healing wounds.
2.2 Factors that affect wound healing and Introduction to chronic wounds
Wound healing is not only a complex process but also very fragile, thus any
abnormality in the wound or body may lead to non-healing (chronic) wounds. Wound healing is affected by various local and systematic factors around the wound/body that
effect the efficacy, speed and manner of wound healing[9, 14]. Local factors are those that directly influence the characteristics of the wound, which include moisture around the wound, edema, ischemia and necrosis, as well as low oxygen tension. Whereas systematic
5
factors stem from the overall health/disease condition of an individual and include
inflammation, malnutrition, metabolic diseases, advanced age, ionizing radiation and
diabetes mellitus[7, 9, 14]. More than 80 % of chronic wounds are associated with venous
insufficiency, high blood pressure or diabetes mellitus and researchers are also of the
opinion that the underlying problems with chronic wounds are bacterial colonization, local
tissue hypoxia repeated ischemia-reperfusion injury and cellular as well as systemic
changes of ageing[9]. Each of these factors individually or in combination may lead to non-
healing wounds. The wounds which take more than 6 weeks to close/heal are called as
chronic wounds[9]. Chronic wounds usually are trapped in one or two phases of wound
healing and do not mature to later stages, leading to non-healing wounds. For example,
most of the chronic wounds become trapped in the self-sustaining inflammatory phase and
leading to improper wound healing[7, 9, 11, 17].
2.3 Importance of oxygen in wound healing
Local tissue oxygen level plays a vital role in wound healing process as this
reparative process needs more oxygen than normal tissue for increased demand for cell
proliferation to replace lost cells, bacterial defense, angiogenesis and collagen deposition[9, 14, 18]. In chronic wounds oxygen partial pressures in subcutaneous tissue may decrease to as low as 5 mm Hg, whereas in normal non-diabetic wounds/tissues it decreases to 30 mm Hg from 40 mmHg in normal tissue/skin[10, 11]. In fact, a reduction in oxygen level may lead to non-healing chronic wounds and studies on ischemic rabbit ear have shown that decreasing PO2 levels from 40-45 mmHg to 28-30 mmHg led to 80 %
deceleration in wound healing[19]. Other studies have also shown that even a moderate
decrease in tissue oxygenation level leads to significant increase in chance of infection,
6 while in vitro studies on neutrophils have shown that below 40 mmHg of PO2 they lose their bacterial elimination function[20]. Oxygen is vital for cell metabolism and energy production as it plays a key role in the conversion of ATP to ADP, providing energy through oxidative phosphorylation, this energy helps in cell migration and replication
(proliferation phase). The production of reactive oxygen species (ROS) such as peroxide
- - - anions (HO2 ), hydroxyl ions (HO ) and superoxide anions (O2 ) in the wound is strictly related to oxygen dependent NADPHlinked oxygenase enzyme catalytic process. [9, 14].
ROS also play a key role in oxidative bacterial elimination and also coregulate vital processes in wound healing such as cytokine release and cell proliferation[9].
2.4 Biomaterials for dermal wound healing:
Biomaterials are classified as synthetic/artificial and natural materials[21, 22].
Biomaterials can form hydrogels, nano fibers, membranes, scaffolds and sponges that can find applications in wound dressing[23, 24]. Synthetic biomaterials dressing include polyurethanes and their derivatives, Teflon and silicone materials, whereas natural biomaterials are derived from cellulose, starch, chitin and chitosan, alginic acid, hyaluronic acid, and collagen[21, 22]. Each have their own advantages and disadvantages in terms of synthesis and usage. There is no single material which is ideally suited as a dressing for all types of wounds. Each type of wound requires a dressing with specific properties, which may not be applicable for other wound types. For example, some wounds may need dressings that absorb more fluids whereas for others wounds may need growth factors.
Synthetic biomaterials can be synthesized and modified in a controlled manner, which results in homogenous and repeatable products with similar physical and chemical properties. They are also stable to heat and light and may exhibit good mechanical strength.
7
These materials are non-immunogenic and non-biodegradable. Some of the examples of
clinical synthetic biomaterial wound dressings are Pellethane®2363-80A, Tegaderm® and
Lyoform®[21]. Natural biomaterials, which are derived from the are more beneficial in terms of immunogenicity, biodegradability, are naturally resistant to foreign bodies and by nature they are more suitable for cell proliferation and other cellular activities exhibiting properties similar to extracellular matrix, but the synthesis and modification of these materials is often laborious and modifying these materials may result in loss of many of
their beneficial properties[21, 22]. Typically, these natural polymers have a downside of exhibiting low mechanical strength, which limits applications. Sterilizing biomaterials by
autoclaving may also lead to lose their properties and synthesis of products with consistent
chemical and physical properties is also difficult. They may be sensitive to heat and light
and may degrade when exposed to them. FIBRACOL® Plus, Promogran Prisma® Matrix,
and Helitene® are some examples of collagen wound dressings are in the market and
Surgifoam®, Gelfoam® are gelatin based hemostatic sponges available in the market.
PolyMem, Restore, SORBSAN are some examples of alginate based wound dressings
available clinically[25]. HemCon® bandage and QuickClot® are two examples of chitosan
based wound dressings.
Biomaterial wound dressings can also incorporate active molecules such as
enzymes, growth factors, antimicrobial agents, antioxidants, hormones, vitamins, and other
agents, which help in accelerating wound healing[21]. Earlier studies on animal wound
healing has shown that supplying growth factors such as epidermal growth factors, fibroblast growth factors and Platelet derived growth factors results in improved wound healing[26-28]. Extensive research is continuing in this field and is in the process of
8
advancing to clinical trials. In conclusion we can say that biomaterials, both synthetic and
natural have many applications in wound dressings and are beneficial in providing and
supporting the environment around the wound to heal.
2.5 Current Therapies to Treat Chronic wounds:
A B
Figure 2-2. A. Hyperbaric Oxygen therapy [3]; B. Negative pressure wound therapy / topical oxygen wound therapy [6]
The current clinically available therapies to treat chronic wounds are hyperbaric oxygen therapy (HBOT) and topical oxygen therapy via tent/bag. In HBOT a patient goes into a 100 % oxygen chamber at 2-3 atm oxygen pressure for a 60-120 minutes session per
day for 5 days a week (Figure 2-2 A) [16, 20, 29]. Generally, 10 to 30 treatments are
performed per patient. Previous studies on diabetic patients with ulcers of the lower legs
(n= 68) treated with HBOT and standard care vs. standard care alone resulted in a significant lower amputation rate in the HBOT group[30]. In another study on ischemic rabbit ear ulcers with either HBOT alone and HBOT in combination with PDGF and TGF-
9
β1 treatment, HBOT alone showed increased production of new granulation tissue[31].
HBOT is widely used to deal with chronic wounds, but this not economical as one hour
HBOT session costs between $165 to $ 250 in private clinics and $2000 in hospitals.
HBOT also requires specialized facilities, which are not easily available. Other complications with HBOT are oxygen toxicity, air trapping inside body parts such as lungs and also leading to rupture of ear drums[9, 15].
Topical oxygen therapy is supplying O2 gas via a tank or small portable O2 gas generator in combination with a tent/bag (Figure 2-2 B)[32]. The advantages of this therapy are low cost, reduced risk of oxygen toxicity and home treatment. Study of TOT on 58 wounds in 32 patients showed that 38 wounds in 15 patients closed completely, but this experiment was conducted without controls[33]. Lack of clinical statistical data of this therapy makes it hard to comment on.
To overcome the short comings of above mentioned therapies to treat chronic wounds, scientists are looking for simple solutions to treat chronic wounds in an economically viable way, easy to apply, easy to remove without pain and convenient to the patient without interrupting their professional and personal life. A bandage type wound dressing that supplies supplemental oxygen could be a better option as it has the ability to heal these chronic wounds in a safe and convenient manner.
10
2.6. Artificial oxygen carriers and oxygen generating biomaterials:
Figure 2-3. Pentadecafluorooctanoyl chloride (Example of PFC)[7]
Artificial oxygen carriers (AOC’s) are materials which have the capability to bind,
transport and unload oxygen in the body, similar to native hemoglobin[34, 35]. They are
also called as oxygen therapeutics by some. Currently the two major types of AOC’s are
hemoglobin based-oxygen carriers (HBOC’s) and Perfluorocarbons (PFCs)[34, 35].
Oxygen generators or oxygen releasing biomaterials (ORBS) are made up of peroxides
embedded into biomaterials[36]. The most common ORBs include sodium per carbonate,
calcium peroxide, magnesium peroxide and hydrogen peroxide incorporated into
biomaterials such as PLGA (Poly(D,L-lactide-co-glycolide). These solid peroxides release oxygen upon exposure to water. Though some in vitro and in vivo studies have shown
positive effects of these oxygen generating materials. The concerns with these materials
are low consistency in oxygen release (oxygen bursts initially), release of free radicals,
change in local pH environment and metal oxide deposits that may create toxicity to cells
and lead to cell death[36].
Hemoglobin solutions show sigmoidal O2 dissociation behavior whereas PFC’s
show linear relationship with PO2 and concentration following Henry’s law[34, 35].
Perfluorocarbon’s (PFCs) can increase O2 solubility in water by 20 fold., from 2.2 mmHg
to 44 mmHg at STP[34, 37, 38]. This property of PFC’s to dissolve high amounts of O2 for
11
delivery to places with low O2 amounts (chronic wounds) is the primary motivation for using PFCs in most clinical applications. The other advantage with PFCs is that it they can also dissolve CO2, CO and N2 gases which makes them useful in not only to supply oxygen
but also dissolving waste gases from human body. The first beneficial effects of these PFCs
were found in 1960 when Clark and Gollan submerged mice in oxygenated liquid PFC
solution showing the animals were able to exchange O2 and CO2 in the liquid[39].
Examples of PFCs have been used clinically, including Fluosal-DA 20% which was used
during cardiac angioplasty in early 1990’s till 1994 when it was taken out of market due to
its storage complications. Another PFC emulsion Oxygent made of 58% perflubron has
also shown interesting results in biocompatibility, oxygen carrying capacity and stability
of the material in phase 1 and phase 2 trails, but it has shown increased stroke risk and
adverse neurological side effects under phase 3 trails. Despite this here is no clear evidence
whether those effects are due to Oxygent or due to aggressive procedures[39, 40]. In
another recent study PFCs encapsulated into alginate beads have improved O2 transport to
human hepatocellular carcinoma (HepG2) cells and resulted in increased cellular metabolic
activity and viability[37]. There is considerable interest in scientists in using PFCs as O2
carriers in a variety of biomedical and bioprocess systems as blood substitutes. Currently,
PFCs have been utilized in many applications, including tissue oxygenation fluids, oxygen
vectors for artificial blood, perfusates for isolated organs, gas carriers in eye surgery,
diagnostic image agents, and drug delivery. The down side of PFC’s is that they are
hydrophobic and not miscible with water. Thus if one wants to use them in aqueous solutions they must be emulsified or made into colloidal suspensions. PFC’s covalently
12 immobilized to biomaterials (like chitosan) have not been reported widely due to the non- availability of non-toxic chemical synthesis methodologies under clinical trials.
2.7. Chitosan and its properties:
Figure 2-4. Structure of chitosan [4]
Chitosan is a partially de-acetylated derivative of chitin. Chitin is the second most abundant natural polysaccharide after cellulose on earth and is composed of β(1→4)-linked
N-acetyl glucosamine [4, 41]. Chitin is the structural element in exoskeleton of crustaceans
(such as crabs and shrimp) and cell walls of fungi[4]. It is a linear polysaccharide consisting of β-(1–4)-linked D-glucosamine residues with a variable number of randomly located N- acetyl glucosamine groups. Chitosan’s molecular weight may range from 10 to 1,000 kDa depending on the source and preparation method. Chitosan is normally insoluble in aqueous solutions above pH 7, but, it can be soluble below pH 5 in acidic solutions like dilute acetic acid. The molecular structure of chitosan is shown in Figure 2-4. Chitosan is biologically renewable, biodegradable, biocompatible, nontoxic, and bio-functional material[4, 42]. It has a hydrophilic surface promoting cell adhesion, proliferation, and differentiation and allows minimal foreign body reaction on implantation. It has many applications due to its structure in biomedical fields including: bone healing treatments, wound healing accelerator, hemostatic agent, antibacterial, antifungal, artificial skin,
13 surgical sutures, artificial blood vessels, controlled drug release[43, 44], contact lens, eye humor fluid, bandages, burn dressing, blood cholesterol control, anti-inflammation, and tumor inhibition[4, 41]. Chitosan by its structure has free amines and hydroxyl groups which can undergo secondary modifications, thus giving birth to modified chitosan polymers which have extended properties that can be useful in biomedical applications[4,
7, 45]. Another important property of chitosan is its biodegradability; it is degradable by hydrolyses, which include the lysozyme enzyme produced in the skin[17, 45]. This property of chitosan along with non-toxic and hemostatic properties make it a good choice for wound healing applications as a hydrogel. Two chitosan based materials are approved by FDA for human use and are commercially available - the Hemcon bandage and Quick clot.
2.8. Hydrogels:
A B
Figure 2-5. A. Sample hydrogel[2] B. Effect of water on hydrogel[8]
Hydrogels are the polymeric materials that can store water in them along all the 3 dimensions. In hydrogels mass fraction of water is very higher than the polymer and more than 90% of the hydrogel is made up of water[46]. Hydrogels have applications in many
14 industrial and environmental fields. Generally, they are prepared by dissolving a hydrophilic polymer material in water and crosslinked to form a gel with the help of a cross linker. Hydrogels also find applications in wound healing due to their properties like:
Maintain moist environment
Absorb the excess exudates from wound bed
Allow gaseous fluid exchange
Protect from bacterial infection
Absorption & retention of Wound caring growth factors and their
bioactivity
Non adherent & easy to remove from wound[8, 47].
These above mentioned properties of hydrogels make them ideal for use as wound dressing material.
2.9. Fluorinated Methyl Acrylamide Chitosan (MACF):
Figure2-6.Example MACF (Chitosan modified with Pentadecafluorooctanoyl chloride (PFC)) [7]
Oxygen carrying PFC’s can be incorporated into chitosan and allowed to form a hydrogel, can be utilized as an oxygen supplying wound dressing to treat chronic wounds.
PFC’s can be incorporated into chitosan by using Schiff base nucleophilic substitution
15
(Figure 2-10). MACF dissolved in water is made into a hydrogel with the help of a photo initiator and UV-light (free radical photo polymerization)[7, 17]. The oxygen loaded by the hydrogel is dependent on both aliphatic or aromatic PFC’s and length of chain of PFC’s.
Studies conducted on 3 different PFCs pentafluoropropionic anhydride (Ali5F), pentadecafluorooctanoyl chloride (Ali15F) and 2,3,4,5,6- pentafluorobenzaldehyde (Ar5F) have shown that MACFs with the most fluorine’s per substitution (Ali15F) showed the greatest uptake and release of oxygen. Interestingly, adding PFC chains with a fluorinated aromatic group (Ar5F) considerably enhanced oxygen uptake and extended release compared with a linear PFC chain with the same number of fluorine molecules (Ali5F)[7].
This phenomenon is expected as in aromatic rings all the fluorine atoms exist in a same plane and attract oxygen molecules with greater force and bind them strongly when compared with aliphatic PFC’s which have fluorine’s oriented in different (all 3 planes), thus attraction or force being distributed to all sides leading to lower oxygen uptake and faster release, Whereas the oxygen uptake and release properties are higher in
MAC(Ali15)F because it has more number of fluorine molecules substituted per PFC.
Thus, naturally it has more oxygen uptake capacity and leading to longer release when compared with other two MACF’s[7].
The ability for significant oxygen uptake and release of fluorinated methylacrylated chitosan motivates us to go forward and study its toxicity and biocompatibility and also to evaluate its benefits for chronic wounds in both in vivo and in vitro.
16
2.10 Introduction to in vitro wound healing assays
Cell migration, cell proliferation, metabolic activity, protein synthesis and adenosine tri phosphate synthesis (ATP) synthesis play a vital role in wound healing process and therefore evaluating the beneficial effects of MACF on these wound healing processes is the primary objective of this study[48, 49]. To evaluate these cellular functions in vitro on human dermal fibroblasts and human epidermal keratinocytes several biomolecular and biochemical assays can be used such as scratch assay, PrestoBlue assay,
Micro BCA assay PicoGreen assay and ATP (Adenosine tri phosphate) determination assay. Here a brief introduction is given to the uses and applications of these assays.
The scratch assay is a straight forward and economical method to study cell migration in vitro. This assay is based on the principle that whenever a gap is created on a confluent cell monolayer, cells on the edges of newly created gap will move towards the gap until new cell-cell interactions are established.[50] This assay mimics the cell migration occurring in vivo and is useful in studying the effects of cell matrix and cell-cell interactions on cell migration. Besides monitoring the migration of homogeneous cell populations, this assay can be used to study the migration of individual cell in the leading edge of the scratch using time lapse microscopy[51].Generally, Cell migration is evaluated by making a scratch on a 100% confluent cell monolayer with the help of pipette tip or other source, which leads to gap between the confluent cells which makes the cells at the edges of scratch to migrate and fill the gaps[50, 52, 53]. To evaluate the scratch closure/cell migration images are taken at regular intervals under the microscope until the scratch closes.
17
PrestoBlue (also known as Almar blue) Cell Viability Reagent is a ready-to-use reagent for rapidly evaluating the viability and proliferation of a wide range of cell types.
PrestoBlue reagent is quickly reduced by metabolically active cells, providing a
quantitative measure of viability and cytotoxicity in very short span of time[54].
“PrestoBlue contains resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide), which is
metabolically reduced to resorufin (7-hydroxy-3H-phenoxazin-3-one) and results in
absorbance shift with proportional to metabolic activity”[55]. So with this assay cells
which are more metabolically active reduce resazurin more and generate greater
fluorescence signal under spectrophotometer.
The micro Bicinchoninic acid (BCA) assay is a colorimetric assay used to quantify the protein concentration in a solution[56, 57]. The total protein content is found by
measuring the color change in the samples from green to purple in proportion to the protein content and evaluating this color change with the help of colorimetric techniques. Color change in samples is due to the reduction of Cu2+ in BCA reagent to Cu- upon interaction
with protein. The applications of Micro BCA assay include protein quantification in protein
purification, electrophoresis, cell biology, molecular biology, and other research
applications[56, 57].
PicoGreen dsDNA Quantitation Reagent is an ultra-sensitive fluorescent nucleic
acid stain for quantitating double-stranded DNA (dsDNA) in molecular biological procedures such as cDNA synthesis for library production and DNA fragment purification for sub cloning, as well as diagnostic applications, such as quantitating DNA amplification products and primer extension assays[58]. Quant-it PicoGreen reagent (Life Technologies) contains fluorescent nucleic acid dye which binds with double stranded ds-DNA and
18
excites upon UV light application and emits light proportional to the concentration of ds-
DNA in the samples. Thus fluorescent values are proportional to the concentration of ds-
DNA and ds-DNA is proportional to the total cell number in the sample[7, 59].
ATP molecules transport energy within a cell for its metabolic activities, thus finding the total ATP content in a cell reveals the total chemical energy within a cell[60].
Total ATP content in cells can be determined by bioluminescence using a luciferase- luciferin system. Luciferin in the presence of luciferase catalyst reacts with ATP and converts it to ADP and produces light[61]. This light emitted is proportional to the ATP content in the cells and measured using a luminometer. The numerous applications of this assay include detecting ATP production in various enzymatic reactions, including ATPase and NADPH oxidase, as well as for detecting low-level bacterial contamination in samples such as blood, milk, urine, soil and sludge. The luciferin – luciferase bioluminescence assay has also been used successfully to study the effects of antibiotics on bacterial populations and to distinguish cytostatic versus cytocidal potential of anticancer drugs on malignant cell growth[62].
In summary, given the importance of oxygen in treating the chronic wounds, the main objective of this in vitro study is to evaluate the beneficial effects of oxygenated
MACF gel in improving vital wound healing cellular processes such as proliferation, metabolism, migration etc. under both normoxia and hypoxia environments and also to evaluate the toxicity and biocompatibility of this material. The MACF hydrogel studied here provides a promising solution in delivering oxygen to the wound, sustaining enhanced local levels of oxygen to overcome hypoxia and providing the beneficial moist environment for enhancing wound reparative processes. Using MACF eliminates the
19 negative effects or concerns related with current oxygen delivering therapies to treat chronic wounds such as hyper baric oxygen therapy and oxygen generators and provides a convenient and inexpensive wound dressing to treat chronic wounds.
20
CHAPTER III
EXPERIMENTAL DESIGN
3.1 Preparation of fluorinated methacrylamide chitosan and methacrylamide chitosan
polymers:
Fluorinated methacrylamide chitosan (MACF) and methacrylamide chitosan
polymers were synthesized and characterized as previously described[7, 17]. Briefly, to
prepare MACF first 3 wt.% chitosan (Mw 190,000-230,000 Da, Sigma-Aldrich, St. Louis,
MO) is dissolved in 2 vol% acetic acid /water. Fluorinated groups were added to the
chitosan by adding 0.14 M pentadecafluoro octanoyl chloride (Sigma-Aldrich, Saint Louis,
MO, USA) and stirred at speed of 60 rpm for 48 hours. Next this solution is modified with
methacrylic anhydride (Sigma-Aldrich) to add methacrylate groups to the polymer (As shown in the figure 3-1A) to result in MACF. To prepare MAC chitosan is modified with
only methacrylic anhydride, so it involves only step 2 in Figure 3-1A[7, 17, 55]. Then this
MACF/MAC solution was placed in dialysis membrane (12,000–14,000 Da molecular
weight cut-off Spectra/ Por, Spectrum Labs, Rancho Dominguez, CA) dialyzed against
deionized water for 3 days with 3 changes of water each day followed by lyophilizing
(freeze drying) (Labconco, Kansas City, MO) to yield dry MAC/MACF
polymer[7].Then1H and 19F NMR (Varian 500 MHz) was conducted to find %
methacrylation and % fluorination respectively as previously described[7]
21
Figure 3-1. A. Reactions involved in the preparation of MACF. Step1: -Addition of PFCs to chitosan. Step2: Methacrylation. B. Figure representing oxygen molecules loaded to the MACF hydrogels by Van der Waal’s forces and reloading it at the necessary places such as hypoxic environments.
3.2 Hydrogel Preparation:
To prepare the hydrogel, first polymer (MAC/MACF) is dissolved at 2.5 w/v% in
ultra-pure water (MilliQ Direct 8 system at 18 M ohm resistance, Millipore, Billerica, MA).
Then it is sterilized by autoclaving (liquid cycle, 10 min per 15 ml of solution at 137oC).
Followed by this, photo initiator solution consisting of 300 mg ml-1 1-hydroxycyclohexyl
22
phenyl ketone (Sigma-Aldrich) in 1-vinyl-2-pyrrolidinone (Sigma-Aldrich) is added to the
polymer solution at 10 µl per g of solution and thoroughly mixed and degassed at 3000
rpm for 2 min (Speed Mixer DAC 150 FVZ, Hauschild Engineering, Hamm, Germany).
Then the hydrogels were made by transferring the solution (300 µL) to 96 well-plate and
exposing them to UV light (365 nm and 16-19 mW/ cm2) for 5 min. Next, they are washed
thoroughly with PBS in 500 ml speed mixing cup with 3 changes per each day for 3 days
to remove all the unreacted polymer and cross linker. The dimensions of the final hydrogels
are approximately 6 mm and in diameter and 10 mm height respectively.
3.3 Saturating MACF hydrogels with oxygen
To load the MACF hydrogels with oxygen first they are soaked in media (Human
keratinocyte media (ATCC)/Human fibroblast media (Life Technologies; Carlsbad, CA,
USA) overnight at 4oC. Then they are loaded with oxygen by supplying the gaseous oxygen
for 30 min at 5 Psi such that the hydrogel is saturated with oxygen. This optimum time to
saturate the gels with oxygen is determined in our lab by finding the oxygen levels in the
gels at regular intervals of 10, 20, 30 and 40 min of oxygenation and found that oxygen
level in gel is saturated at 30 min and didn’t change significantly from there (Supporting
data can be found in Fig. S1 in Appendix)
23
3.4 Effect of Oxygenated MACF gel on cell media
To find the effect of oxygenated MACF gels in supplying additional oxygen to media, firstly MACF hydrogels were made as described in Section 3.3 and they were oxygenated as in Section 3.4. Then they are transferred to the top of 1 ml media in 24 well-plate by keeping them in inserts (6.5 mm transwell with 8.0 µm pore polycarbonate membrane insert, sterile, corning, NY, USA) and these experimental well plates are maintained in a normoxic incubator at 37oC.Next, the oxygen level in the media is monitored with the dot type oxygen measuring sensors (FireSting O2, Pyro Science, Aachen, Germany) connected to a fiber-optic oxygen meter(Pyro science) by the bare optical fiber (Pyro Science) at t =
0 ,2, 7, 20, 24 and 48 h and recorded. To compare, oxygen level in the only media is also monitored at the similar time points and recorded.
3.5 Cell culture
Human dermal fibroblasts used for these studies were harvested from human neonatal foreskins. These fibroblasts were expanded in Dulbecco’s modified eagle medium
(DMEM) containing 10% fetal bovine serum (FBS) and 100 μg/mL penicillin−streptomycin (all Life Technologies; Carlsbad, CA, USA).
Human epidermal keratinocytes neonatal (HEKn) used for these studies were obtained from ATCC and expanded in dermal cell basal medium containing keratinocyte growth kit along with penicillin-streptomycin- amphotericin and phenol red (all are obtained from ATCC). HEKn were expanded and passaged according to the standard protocol given by the company.
24
Both these cells were expanded, passaged and grown in normoxic incubator (21%
o O2,5% CO2 and 74% N2 at 37 C). A hypoxic environment (1% O2,5% CO2 and 94% N2 at
37oC) was required for studies and created by the trigas incubator (Forma Scientific water
jacketed incubator, Model no 3159). The trigas incubator is purged with excess nitrogen
and CO2 to remove excess O2 beyond 1% and to make the environment required for
hypoxic experiments (1% O2). Media used for growing the hypoxic cells is equilibrated to
hypoxic environment prior to use, by maintaining the media in T75 flasks for 24 hours in
hypoxic environment (hypoxic incubator)[63].
3.6 Cell proliferation studies
Human cells (neonatal human epidermal keratinocytes and neonatal human dermal
fibroblasts) were seeded in 24 well-plates at 8,000 cells/cm2 and allowed to attach and
spread along the surface overnight at 37oC in a normoxic environment. Thereafter,
normoxic wells were continued in the normoxic incubator with normoxic media. Chronic
(hypoxic) wells were transferred to a hypoxic incubator (1% O2, 5% CO2 and 94% N2 at
37oC) and media on the chronic cells is also the media equilibrated with the hypoxic
environment[63]. The following day, the treatments oxygen saturated MACF and MAC hydrogels were applied to wells in both normoxic and hypoxic environments alongside the
no treatment wells that were also maintained in both the environments to compare with the
treatments. Cells were allowed to proliferate for 3 d (72 h). Thereafter, images were taken
using an optical microscope to evaluate the proliferation of the cells under each treatment
by observing their morphology. To evaluate the effect of oxygenated MACF gel on human
25
cells proliferation its morphology, metabolism, synthesis, total double stranded
deoxyribonucleic acid (ds-DNA) and adenosine triphosphate (ATP) quantification were
evaluated for each treatment using respective assays[59, 61].
3.6.1 Presto blue to evaluate cell metabolism (viability)
To evaluate the cell metabolism (viability), first 1 ml of 10 % Prestoblue (Life
Technologies, Temecula, CA) in serum free growth media is applied on the cells and then allowed to incubate for 90 min at 37oC. Next supernatant solution from each well is
transferred to 3 wells (replicates n=3) of 96 black well plate (Greiner). “The Presto Blue
solution contains resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide), which is
metabolically reduced to resorufin (7-hydroxy-3H-phenoxazin-3-one) and results in an
absorbance shift with proportional to the metabolic rate”[59]. Thereafter its fluorescence
values at excitation wavelength of 560 nm and emission of 590 nm were determined with
the help of spectrophotometer (Infinite M200, Tecan, Grödig, Austria) and compared
against DTT standard curve to get the absolute values of cell viability in terms of equivalent
DTT concentration[59].
3.6.2 Digesting the cells in lysozyme
Adherent cells in 24 well plates were digested in 1 ml of 25 mg/ml lysozyme
(Sigma) dissolved in PBS at pH 5 for 24 h at 60oC with through mixing by pipetting once every 3 to 4 h[59]. Thereafter, the lysozyme solution containing digested cells were transferred to 2 ml tubes and stored at 4 oC for the assays below.
26
3.6.3 Micro BCA assay to evaluate protein synthesis
Total protein was quantified by using the Micro BCA protein assay kit (Thermo
Fisher Scientific). The micro BCA assay is performed to determine the total protein content
according to the manufacturer’s protocol. Briefly, first digest samples were diluted in PBS
(15 µl of sample is added to 1000 µl of PBS). Thereafter 150 µl of each diluted digest sample is added to the clear 96 well plate followed by 150 µl of micro BCA reagent. After this they are incubated at 37oC for 2 hours and absorbance values were determined under
spectrophotometer. A standard curve is created with bovine serum albumin (BSA; VWR,
Radnor, PA, USA) diluted in PBS against absorbance values to provide standards. Absolute values of the protein for each well were determined with the help of the standard curve[59].
3.6.4 PicoGreen assay to determine total double stranded DNA
To determine the total no of cells in each well, total ds-DNA is quantified by using
the Quant-iT PicoGreen dsDNA kit (Life Technologies). To determine the total ds-DNA
in each well, first digested samples were diluted (100 µL of digested sample in 1000 µl of
PBS). Next, 150 µl of each diluted digest sample were added to the black 96 well-plate in
triplicate followed by 150 µl of Quant-it PicoGreen reagent and incubated for 10 min at
room temperature in the dark[7]. Next, fluorescence values were determined under
spectrophotometer (Tecan Infinite M200). Observed fluorescence values were compared
against standard curve created using Quant-iT Pico Green ƛDNA standards to find the total
dsDNA concentration in each well[7].
27
3.6.5 Adenosine triphosphate (ATP) quantification
ATP quantification in each well was determined by using a bio luminescence ATP determination kit (Thermo Fisher Scientific, A22066). The assay employs luciferin to reacts with ATP in the presence of luciferase, which in turn is converted to oxyluciferin, emits light in proportional to the amount of ATP in the cells[61]. First the standard reaction solution is prepared by mixing the all the components namely, 20X TE buffer, dH2O, DTT,
D-Luciferin and firefly luciferase in appropriate quantities as stated in the company’s protocol. ATP standard solutions were prepared by diluting the ATP in dH2O and the ATP standard reaction solution. Thereafter, 500 µL of above standard reaction solution is added to each sample in a clear 24 well-plate and luminescence values were recorded for unknown wells and the ATP standard solutions. Finally, the absolute ATP values of the unknown samples was found by comparison with known values from the standard curve.
28
3.7 Scratch assay to study cell migration
Figure 3-2. Schematic diagram representing steps involved in scratch assay.
To study the effect of supplying additional oxygen using MACF gels on cell
migration, an in vitro scratch assay was conducted on human dermal fibroblasts and human
epidermal keratinocytes (Figure 3-2). For this first, 24 well-tissue culture plates were
coated with 1 ml of sterile 0.2 mg/ml collagen type I in 1X PBS for 2 h at 37oC and then
washed with sterile PBS once[50]. After this, these wells were seeded with 40,000 cells/
cm2 human skin cells (neonatal human dermal fibroblasts or human epidermal keratinocytes)[50, 53]. Then after two days of culture in normoxia or hypoxia, confluent
cells were scraped in the middle of the well along the diameter to create a scratch. Wells
were washed one time with media to remove all the debris created in the well due to scratch.
Next fresh media is added to the wells and cells were allowed to migrate[52, 64]. Images of the scratches were taken at t=0 and t= 24 hours respectively under optical microscope
29
(Olympus IX-81, Tokyo, Japan). Recorded images were used to determine the width of the
scratch using image J (National Institutes of Health, Bethesda, Maryland, USA) and the
resulting percentage of cell migration is calculated for each treatment: percentage of cell migration = Wi-Wf/ Wi *100. Where Wi= width of scratch at t= 0 h and Wf = width of
scratch at T= 24 h.
3.8 Immunohistochemistry procedures
To find the presence of Ki67 protein (antigen Ki-67 is a nuclear protein that is associated
with and may be necessary for cellular proliferation), immunohistochemistry was
performed. For this after the completion of scratch assay, all the cells were fixed in 3.7 %
paraformaldehyde (Sigma-Aldrich) for 30 min and then washed with PBS thoroughly for
3 times. Next, scratch assay wells were washed with PBS for three times and then wells were incubated with the primary antibody (anti ki67 antibody, rabbit monoclonal, abcam,
Cambridge, MA, USA) overnight at 4 oC. Then they were washed with PBS for 3 times
then incubated with the secondary antibody (goat anti-rabbit, Alexaflour488, Life
Technologies) for 1 h in dark at room temperature followed by washing with PBS. Next samples were incubated for 10 min in the dark at room temperature with nuclear Hoechst
(Hoechst 33342, Thermo Scientific) followed by thorough washing with PBS. Finally, sample were mounted using Prolong gold (Thermo Scientific) anti-fade solution.
Fluorescence images were obtained with a fluorescent microscope (OlympusIX-81)
30
CHAPTER IV
RESULTS
MAC and MACF were successfully synthesized and purified. Fluorine (19F) NMR spectra and proton (1H ) NMR spectra of synthesized MACF polymer are presented in
Figure 4-1. 19F NMR showed 38 to 42% PFC substitution and 1H NMR showed 19 to 23
% degree of substitution for methacrylation as previously reported[7].
A
B
Figure 4-1. A. High resolution 19F spectra for MACF and B. High resolution 1H spectra for MACF
31
4.1 Effect of oxygenated MACF gel on cell media:
Figure 4-2. Oxygenated MACF gels elevate PO2 level in culture media. PO2 level is measured in the media with oxygentaed MACF gel as compared with MAC and no gel media at time several time points up to 48 h. Mean ± SD with n=4
The PO2 level in the media with oxygenated MACF gel, MAC gel and their comparison with only media are shown in Figure 4-2. PO2 levels are elevated in media with oxygenated MACF gel for all the time points measured, with the highest value observed at 2 h. The PO2 levels in media with MAC gels are slightly higher than the no media and less than MACF exposed media at all time points.
32
4.2 Effect of oxygenated MACF gels on cell morphology, metabolism and protein synthesis
A D
No gel No
B E MACC
C F
MACF(O)
Figure 4-3: Effect of MACF on human dermal fibroblast morphology. Representative phase contrast images of human dermal fibroblasts grown under different treatments for 72 h. A, B, C are in normoxia (21% O2) and D, E, F are in hypoxia (1% O2). Scale bar is 200 µm
33
A D No gel No
B E MAC
C F
MACF(O)
Figure 4-4: Effect of MACF on human epidermal keratinocytes morphology. Representative phase contrast images of human dermal fibroblasts grown under different treatments for 72 h. A, B, C are in normoxia (21% O2) and D, E, F are in hypoxia (1% O2). Scale bar is 200 µm
34
A B
Figure 4-5. The effect of MACF on cell metabolism, as determined by the PrestoBlue asay. MACF improved cell metabolism (viability) under both hypoxic and normoxic environment, as showed by increased reducing power per well (equvalent DTT concentration). A. Human dermal fibroblasts and B. Human epidermal keratinocytes. White bars represent normoxic environment and black bars represent hypoxic environment. Significant difference at p < 0.0001. Letters are significantly different from one another by single-factor ANOVA (p < 0.0001). All data n = 3-4, mean ± SD
Both human dermal fibroblasts and human epidermal keratinocytes exhibited regular
morphology they were uniform throughout the surface as in representative cell morphology
images (Figs. 4-3, 4-4). Further, both human dermal fibroblasts and human epidermal
keratinocytes were increasingly active in the presence of MACF hydrogels under both normoxic and hypoxic environments (Fig. 4.5). Additionally, these results show that overall cell metabolic activity (viability) decreased in low oxygen concentrations
(hypoxia), however, cells regained their metabolic activity upon the application of supplemental local oxygen through our MACF gels.
Supplying oxygen through MACF gels didn’t significantly alter the total protein content in both human dermal fibroblasts and human epidermal keratinocytes under both normoxic and hypoxic environments and were same as no gel treatment controls. In human dermal fibroblasts the total protein content as measured in terms of BSA concentration
35
(µg/ml) were 261.96 ±5.51 µg/ml, 265.39 ±16.18 µg/ml and 270.95 ± 13.5 µg/ml under normoxia with no gel, MAC and MACF treatments respectively. In hypoxia total protein was 239.02 ± 21.48 µg/ml, 248.94 ± 12.77 µg/ml and 266.30 ± 4.7 µg/ml with no gel,
MAC and MACF treatments respectively. Next in human epidermal keratinocytes measured total protein was 269.62 ± 48.76 µg/ml, 273.92 ± 20.41 µg/ml and 287.73 ±
25.56 µg/ml under normoxia with no gel, MAC and MACF treatments respectively.
Whereas as in hypoxia they are 283.42 ± 15.96 µg/ml, 290.09± 7.12 µg/ml and 293.58 ±
12.29 µg/ml with no gel, MAC and MACF treatments respectively.
36
4.3 Effect of oxygenated MACF on total double stranded DNA and ATP levels
A B
C D
Figure 4-6. The effects of MACF on total DNA and total ATP levels. MACF increased total DNA level and total ATP level per well under both hypoxic and normoxic environments. A, C are human dermal fibroblasts and B, D human epidermal keratinocytes. A, B represent total DNA levels measure by PicoGreen assay and C, D represent total ATP level measured via luminescent assay (note different y axis scales). White bars represent normoxic environment and black bars represent hypoxic environment. Letters are significantly different from one another by single-factor ANOVA (p A, C < 0.005; B, D p < 0.0001. All data n = 3-4, mean ± SD
In both cell types (human dermal fibroblasts and human epidermal keratinocytes) supplying oxygen through MACF hydrogels increased total double stranded DNA in both normoxic and hypoxic environments (Fig. 4-6 A, B), demonstrating cell proliferation. In the same way MACF increased ATP levels in both normoxic and hypoxic environments
(Fig. 4.6 C, D).
37
4.4 Effect of oxygenated MACF on cell migration
A B
Figure 4-7. Oxygenated MACF accelerated cell migration under hypoxia in both human dermal fibroblasts and human epidermal keratinocytes. A. Human dermal fibroblasts B. Human epidermal keratinocytes. White bars represent normoxic environment and black bars represent hypoxic environment. Letters are significantly different from one another by one-factor ANOVA (p < 0.0001). All data n = 3-4, means ± SD
Oxygenated MACF gels reversed some of the impaired cell migration under
hypoxia in both human dermal fibroblasts and human epidermal keratinocytes as shown in
Figure 4-7. By 24 hours scratch closure is complete in all the treatments under normoxia
in both the human skin cell types, whereas in hypoxia they are not closed completely as
shown in representative phase contrast images (Figs. 4-8, 4-9).
38
A
B E
No gel No
C F
MAC
D G
MACF
Figure 4-8. Effect of MACF on human dermal fibroblast migration. Cell migration is studied by in vitro scratch assay. Representative phase contrast images of scratch assay A. Scratch at t=0 for all controls; B-G images of scratch at t= 24 h; B, C, D are in normoxia (21% O2) and E, F, G are in hypoxia (1% O2). Scratch is completely closed in normoxic controls by 24 h whereas as in hypoxic controls it is not completely closed with MACF showing improved human dermal fibroblast migration. Scale bar is 200 µm. 39
A
B E
No gel No
C F
MAC
D G
MACF
Figure 4-9. Effect of MACF on Human epidermal keratinocytes migration. Cell migration is studied by in vitro scratch assay. A. Scratch at T=0 for all controls; B -G images of scratch at t= 24 h; B, C, D are in normoxia (21% O2) and E, F, G are in hypoxia (1% O2). Scratch is completely closed in normoxic controls by 24 h whereas as in hypoxic controls it is not completely closed with MACF showing improved human epidermal keratinocytes migration. Scale bar is 200 µm.
40
4.5 Ki67 IHC staining (Protein embedded with cellular proliferation)
A B
C D
Figure 4-10. IHC staining for Ki67 protein, which is a cell marker for cell proliferation on human dermal fibroblasts. Representative fluorescent images; A, C are images of scratch under Normoxia whereas B, D are under hypoxia. A, B are images taken on the scratch whereas C, D are taken outside the scratch. Scale bar is 200 microns
A B
C D
Figure 4-11. IHC staining for Ki67 protein, which is a cell marker for cell proliferation on human epidermal keratinocytes. Representative fluorescent images; A, C are images of scratch under Normoxia whereas B, D are under hypoxia. A, B are images taken on the scratch whereas C, D are taken outside the scratch. Scale bar is 200 microns
41
IHC images of both keratinocytes and fibroblasts under both hypoxia and normoxia look similar and green fluorescence. Importantly, Ki67 is distributed equally all over the well and not concentrated on the scratch borders (Figs. 4-10, 4-11).
42
CHAPTER V
DISCUSSION
Fibroblasts and keratinocytes are the major cell types present in dermis and
epidermis of the human skin, performing vital cellular wound healing functions, including
cell migration and proliferation [48, 49]. Based on this importance, human dermal
fibroblasts and human epidermal keratinocytes were chosen for the presented studies.
Impairment of wound healing cell functions is one root cause for non-healing chronic
wounds, leading to poor neo-vascularization, granulation and re-epithelization.
Experimental evidence supports that one underlying reason for impaired cellular functions in chronic wounds is a basic lack of oxygen[9, 14, 36, 48]. Thus, supplying sufficient
oxygen directly to chronic wounds could improve wound healing, which is supported by
current clinical practice employing hyperbaric oxygen therapy[65, 66]. Oxygenated MACF
hydrogels provide a potential alternative to locally supply oxygen to wounds, with the
added benefits of a hydrogel (e.g., moist wound healing)[67]. Previous studies the Leipzig
lab have conducted on MACF have shown that a PFC incorporated chitosan polymer is
able to be loaded with oxygen repeatedly and supply it as necessary[7]. Also, these
previous studies have shown that grafting PFCs with a higher number of fluorine’s per
chain (pentadecafluorooctanoyl chloride) provides more benefits when compared with
PFCs chains with less numbers of fluorine’s (pentafluoropropionic anhydride and
43
2,3,4,5,6-pentafluorobenzaldehyde). Thus MACF incorporating longer PFC chains was used in the cellular investigations of this thesis in alignment with prior findings[7].
The results of this study once again support previous reports [7] that MACF
hydrogels maintain a higher than ambient oxygen concentration at equilibrium . From
Figure 4-2 it also seen that the oxygen level in the media with MACF gel is always higher
than the control throughout the period of the experiment and reaches an equilibrium that is
15-17 mmHg above the media only value. Previous in vivo studies demonstrate that in
acute and chronic wounds increment in oxygen level by 5 to 10 mm Hg can enhance wound
healing responses [17], suggesting that MACFs equilibrium oxygenation is in the
beneficial range. Additionally, in both human dermal fibroblasts and human epidermal
keratinocytes the base material MAC has shown improved results in hypoxia when
compared with no gel treatments. This may be due to the oxygen present in the outer layer
of MAC as they are stored in PBS prior to use.
Overall this study revealed that oxygenated MACF gels across the board improved
cellular functions under hypoxia (1% O2) as evidenced by improved in vitro migration and
proliferation of human dermal fibroblasts and human epidermal keratinocytes. Hypoxia is
created to mimic the environment in a chronic wound to study impaired cellular processes
in vitro. Here in our experiments to better replicate the chronic wound hypoxic
environment in vitro, human skin cells were kept in hypoxia for 24 h prior to applying any treatments such as oxygenated MACF to them [63, 68]. Oxygen levels lower than 5% are
generally considered hypoxic but in in vitro studies oxygen levels between 2% and 5% do
not show any significant differences in cellular functions such as proliferation and ATP
synthesis when compared with 21% O2. Whereas, oxygen levels 2% and lower show
44
significantly decelerated cellular functions such as proliferation and ATP synthesis with more deceleration at 1% O2[61]. Further, 0.5% O2 level leads to complete resting of cells thus less utilization of cellular ATP resulting in higher values experimentally[61]. Based on these reports, a condition of 1% oxygen was chosen to perform in vitro studies to best mimic the hypoxic conditions that are encountered with decelerated wound cellular functions. To further recapitulate chronic hypoxic stress on man skin cells, three experimental protocol improvements were also employed: 1. using hypoxia equilibrated media for hypoxia cell culture, 2. maintaining cells for 24 h in hypoxia prior to application of treatments/experiments 3. exposure to moderate hypoxia (1% O2). Results show the impact of hypoxia on cell functions such as migration (Fig. 4-7) and proliferation (Fig.4-6
A, B) to both human dermal fibroblasts and epidermal keratinocytes. Further, metabolism/metabolic activity of the cells quantified using the PrestoBlue assay is lower under hypoxic conditions compared to normoxic conditions in both cell types (Fig. 4-5).
These results were expected as they have been reported in similar hypoxia studies[61, 68,
69]. Most importantly, hypoxic decreases to cell functions were increased with the application of oxygenated MACF gels, demonstrating that MACF has the potential to overcome impaired cellular processes in hypoxia.
Energy for the bulk of cellular functions is provided by ATP. Interestingly, assaying
ATP levels in our experiments shows that MACF treatments enhance ATP in cells (Fig. 4-
6 C, D), providing cells with higher energy to perform their functions even in a hypoxic environment. It is known from the literature that hypoxia leads to decreased oxidative phosphorylation. leading to lowered ATP production in cells, which in turn leads to deceleration of the cellular functions like proliferation and migration[61, 70]. Here in our
45
studies hypoxia reduced ATP levels in human dermal fibroblasts and human epidermal keratinocytes to 30% and 70 % of their original values respectively. However, upon
application of MACF gels, ATP levels again increased and recovered to 72 % and 110 %
(fibroblasts, keratinocytes, respectively) of their original values tells us how beneficial is
this oxygen supplied through MACF gels. IHC images (Figs. 4-10, 4-11) stained for Ki67,
a cellular marker for proliferation. Result show uniform green fluorescence all over the
well, suggesting migration in scratch closure is not due to proliferation.
Comparing oxygenated MACF gels to other oxygen generating biomaterials like
calcium peroxide[71], hydrogen peroxide[72] and sodium per carbonate[73] encapsulated
3D scaffolds are inconsistent in producing oxygen. They also have the downside of also
changing the local pH level while also directly producing reactive oxygen species, all of
which may lead to further death of cells[18]. These oxygen generating biomaterials cannot
be reloaded with oxygen, whereas MACF gels can be reloaded with oxygen as long as they
are present[7].
Overall, studies to date continue to show that MACF is beneficial in improving
wound healing functions via supply of supplemental oxygen from a hydrogel [7, 17].
Studies have also shown that this MACF material is bio degradable[17] and most of the
concerns with PFC’s are related to environmental issues not bio medical. Fluosal-DA 20%
was used during cardiac angioplasty till 1994 but it is revoked from the market basically
due to its storage issues not due to its toxicity issues[39]. It is also important to note that
most concerns with the PFCs are due to its application intravenously at high dosages and
they are reversible and doesn’t result in permanent tissue alteration[40, 74]. But here our
MACF is applied topically that to on the limited area of the wound which potentially limits
46
its systemic interactions resulting in any health issues. Additionally, PFCs incorporated into MACF are not long chain PFCs and whereas PFCs elimination through lungs by the reticuloendothelial macrophages is inversely proportional to its molecular weight (Length of chain) which tells us that PFCs incorporated into MACF which are small chain and in less dosages can be eliminated easily by RES from the lungs[40]. In terms of disposal, the conjugation chemistry to chitosan polysaccharide chains stabilizes the PFCs substantially and greatly reduces lipophilic nature and environmental concerns. Ultimately, our lab will perform the proper safety and cytotoxicity studies as required to gain FDA approval, but we do not foresee any issue with this at this point due to the many beneficial results we have seen till date.
47
CHAPTER VI
CONCLUSIONS and FUTURE WORK
The main goal of these in vitro studies was to evaluate the beneficial effects of
supplying oxygen through fluorinated methacrylamide chitosan for improving cellular
wound healing processes. For this study, two human skin cell types, human dermal
fibroblasts and human epidermal keratinocytes, were chosen and studies were conducted
under both under normoxic (21% O2) and hypoxic (1% O2) conditions. These conditions
best mimic the local environments of acute wounds and chronic wounds respectively. As
stated in the Specific Aims, the overall hypothesis of this study was that an underlying
cause for impaired wound healing in chronic wounds is low tissue oxygenation levels and that supplying topical oxygen via application of oxygen loaded MACF hydrogels can
improve this stalled wound healing process at a cellular level.
In this thesis it is once again revealed that MACF can be saturated with oxygen and
release this oxygen to local surroundings for a prolonged time, while supporting an
equilibrium oxygen concentration long-term that is higher than ambient (Fig. 4.2). Then,
the vital wound healing cellular processes, namely cell proliferation (Fig. 4.6 A, B) and
cell migration (Fig 4.7) that were impaired under hypoxic conditions in both human skin
cell types, were recovered with the application of oxygenated MACF hydrogels.
Cell ATP synthesis, which is reduced under hypoxic conditions by nearly 70 %, also
recovered markedly with oxygenated MACF treatment (Fig. 4.6 C, D). Finally, cellular
48
metabolic activity also increased in the presence of MACF hydrogels as compared to
controls (Fig. 4.7). Overall, my thesis confirmed previous work[7, 17] that this chitosan material incorporated with PFCs is nontoxic, biocompatible and could provide a simple solution for treating chronic wounds to supply oxygen topically via a hydrogel.
Based on earlier in vivo studies[17] and my in vitro findings, further studies can be undertaken both in vitro and in vivo to continue to develop this MACF hydrogel approach for treating chronic wounds. For in vitro directions, as I conducted studies on healthy human skin cells (neonatal derived), further studies can be conducted on skin cells collected from diabetic patients at advanced ages (> 60 years), representing cells sampled from groups that have high chronic wound prevalence. From the literature, it is known that wound healing cellular process such as proliferation and migration are impaired in both these patients’ skin cells and applying our oxygenated MACF gel may enhance these stalled wound healing processes, similar to my studies. Additional in vitro studies could also be conducted on healthy human skin cells (both human dermal fibroblasts and human epidermal keratinocytes) by exposing them to prolonged hypoxic conditions (cultured in hypoxia for at least 6 passages)[63]. Previous studies have revealed that prolonged exposure to hypoxia can impair cell proliferation and metabolism, and these conditions may more accurately mimic chronic wounds. It would be important to determine if similar results are obtained as were found in this thesis, that further impaired cellular processes from prolonged hypoxia can also be recovered upon oxygenated MACF hydrogel treatment.
49
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56 APPENDIX
Figure S1. Data representing saturation time for oxygenating MACF gels. MACF gels were oxygenated for different time points at 5 psi and the oxygen level in the gel is measured with the needle type O2 sensor (FireSting). Acknowledgement: This data was collected by Pritam Patil.
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