Development of cell sheet constructs for layer by layer tissue engineering using the blood vessel as an experimental model 4

Conjugation of a wide variety of biomolecules to blood contacting surfaces has been studied in vascular tissue engineering, which can be broadly classified under three categories: adhesi

unpaired t-test) (Figure 5-2) After repeating the grafting process, water contact angle was found to drop further to 52.0 ± 1.3°, in line with the above observation (p<0.001)

No further improvement could be seen after two rounds of grafting

Water Contact Angle

Figure 5-2: Surface wettability of modified films

Static water contact angle showed a decrease after one round of pAAc grafting, with another significant decrease with a second round of pAAc grafting (** denotes statistically significant difference, p<0.001) No significant change was detected following subsequent grafting (p>0.05)

5.2.1.3 Quantification of carboxyl density

I used a TBO assay to quantify the amount of carboxyl groups on the surface PAAc was grafted onto PCL film surfaces by plasma immobilisation with a 13-fold increase

in surface carboxyl density using Toluidine BlueO (TBO) assay (p<0.01) (Figure 5-3)

**

**

Biaxial stretching is known to influence the morphology and crystal structure of PCL films, due to fibriallar orientation (Ng 2000; Tiaw 2007) Thus, in a concurrent experiment, I studied the effects of biaxial drawing on plasma immobilisation yields

of PAAc A greater grafting yield on biaxially stretched films compared to unstretched / native films, with 60% higher carboxyl density after two rounds of

grafting (242.4±9.3 vs 153.7±10.6 nmol/cm2, p<0.01, TBO method, Figure 3a)

These results suggest that bi-axial stretching breaks down the lamellae structure, leading to increased exposure of more polyester linkages for the facilitation of plasma cross-linking

Number of Repeat Graftings

Figure 5-3: Surface carboxyl density of modified films

Quantification of COOH surface density using Toluidine Blue O Assay demonstrated

a progressive increase in carboxyl density with repeated grafting up to two times, with stretched films incorporating more carboxyl groups than non-stretched films up to three repeated treatments (* denotes statistically significant difference, p<0.01)

5.2.1.4 Assessment of tensile properties

The choice of the plasma-based method to modify the µXPCL surface was made on the basis of its advantageous low depth of penetration However, bioresorbable materials are by nature highly sensitive to degradation effects This issue is compounded in the use of microthin films Moreover, repeat plasma grafting was used that may have compromised the properties further In addition, the modified films were designed to support conjugation of biomolecules, which necessitated the use of carbodiimide chemistry Carbodiimides are commonly used as cross-linkers to stabilise collagen structures (Nam 2008), and may influence the mechanical behaviour

of the microthin film

Thus, to evaluate the extent of mechanical damage, I carried out tensile tests to evaluate the strength of the film following modifications No significant drop was observed as a result of plasma treatment, nor was their any trend to suggest mechanical compromise, irrespective of the number of rounds of PAAc grafting In light of the results thus far, subsequent experimentation was conducted on µXPCL grafted with two layers of PAAc (Henceforth referred to as PCL-PAAc)

Surprisingly, a drop in ultimate tensile strength, albeit insignificant, was observed for carbodiimide treated films It suggests that some form of degradation may have taken

5 10 15 20 25 30 35 40 45

Figure 5-4: Mechanical properties of modified films

Tensile testing of µXPCL-PAAc films demonstrate no trend nor significant difference

in (a) ultimate tensile strength and (b) yield strength as a result of plasma modification (p>0.05) A drop in tensile properties occurred as a result of the carbodiimide activation process, but the decrease was not statistically significant

5.2.1.5 Plasma immoblisation of Polyethyleneimine (PEI)

It is possible to confer greater functionality to the film surface by varying the nature

of the grafted polymer This includes polyols for the introduction of chemically useful hydroxyl groups (Bures 2001) or even polymers with specific configurations, such as star-shaped dendrimers (Won 2003) Here, I studied the immobilisation of PEI to provide amine groups

PEI was successfully grafted using a similar process However, because of the lack of reliability of amine quantification techniques, I analysed the PEI modified surface

with XPS Unlike PCL and PAAc, PEI contains nitrogen, and will show up on the XPS scan through the introduction of the N1s peak (Figure 5-5)

0 200

400 600

800 1000

Figure 5-5: XPS survey of PEI-immobilized film

XPS wide survey spectra of PEI grafted µXPCL films shows the introduction of an N1s peak due to the presence of amines

5.3 Conjugation of Biomolecules

In this section, I investigated the possibility of conjugating bioactive molecules onto the modified films for possible tissue engineered blood vessel applications Conjugation of a wide variety of biomolecules to blood contacting surfaces has been studied in vascular tissue engineering, which can be broadly classified under three categories: adhesion proteins, anticoagulants and antibodies I had chosen a candidate

5.3.1 Conjugation Of Heparin

The conjugation of heparin onto blood contacting surfaces has been well-studied Beyond anti-coagulatory properties, heparin has been shown to possess growth factor binding sites and has been shown to improve the efficacy of growth factors (Mitsi 2008) It has also been shown to reduce complement activation (Girardi 2005) Moreover, heparin-like proteoglycans are found abundantly on basement membranes and on the surface of EC, suggesting that heparin will be suitable for lining blood contacting surfaces

In my initial experiments with grafting heparin onto PCL-PAAc, low and inconsistent engraftment yields were observed This may have arisen due to inefficiencies in the deprotonation of the heparin sodium salt Consequently, I opted to graft heparin onto the PEI modified PCL instead XPS scans reveal the introduction of an S2p peak due

to the incorporation of the highly sulphated heparin Presence of the N1s peak was due to the PEI

0 200

400 600

800 1000

Figure 5-6: XPS survey of heparin conjugated PCL-PEI film

XPS wide survey spectra of modified film following conjugation of heparin,

5.3.2 Conjugation Of Collagen

Collagen is commonly used for surface modification in tissue engineering applications to improve cellular adhesion Collagen contains RGD (Arg-Gly-Asp) sites that are recognised by cells via integrins I proceeded to conjugate collagen onto PCL-PAAc films by carbodiimide chemistry XPS scans demonstrate that collagen was successfully grafted onto the film surface, as evidenced by the N1s peak However, it was also noted that significant contaminants were present The source of collagen used varies greatly from batch to batch due to its direct isolation from animal products (oxen tails), and may have contributed to the contamination observed here

0 200

400 600

800 1000

5.3.3 Conjugation Of Fluorescent Antibodies

To visualise the distribution of the conjugate, I proceeded to graft fluorescently labelled biomolecules on the surface Commercial fluorescently labelled antibodies were used because they are consistent, well-characterised and optimised to reduce signal-to-noise ratio The antibodies were grafted on successfully using carbodiimide chemistry In contrast, without carbodiimide conjugation, nonspecific adsorption occurred, resulting in the diffuse staining Following washing for 72 hours under representative physiological conditions (mechanical shaking in physiological saline at body temperature), most of the antibodies on the untreated film were washed off, leaving some residual adsorbed antibodies Labelled activated films continued to express fluorescence

Figure 5-8: Micrographs of fluorescent-label conjugated PCL-PAAc

Epifluorescent micrographs of films conjugated with Alexafluo 488 goat anti-mouse

secondary antibody The label was conjugated onto activated films, and fluorescence

5.3.4 Conjugation Of Monoclonal CD34 Antibody

Antibodies have previously been proposed for use as “capture surfaces” in blood

renewing passivated surface More recently, antibodies against specific cell types have been proposed In particular, surfaces immobilised with antibodies against CD34 have been studied for recruitment of circulating endothelial progenitor cells This represents a viable option for vascular graft design

5.3.4.1 XPS analysis of PCL-CD34

CD34 antibodies were grafted onto PCL-PAAc surfaces (PCL-CD34), and verified by XPS (Figure 5-9) Wide survey XPS scans show increased O:C ratio following PAAc grafting, and then the introduction of an N1speak following the conjugation of the CD34 antibody, indicating presence of the protein High resolution surveys of the C1s core spectra revealed the presence of peptide bonds I also noted an increase in the percentage of carboxyl containing groups on PCL-PAAc, as compared to µXPCL This dropped following the conjugation of CD34 antibody, as the carboxyl groups were consumed in the conjugation process

Figure 5-9 XPS analysis of PCL-CD34

Engraftment of CD34 antibody a) XPS wide survey spectra of the modified films demonstrating successful engraftment (b) Relative intensity of the deconvoluted C1S spectra of µXPCL films shows increase in carboxyl groups following PAAc engraftment,followed by introduction of peptide groups following CD34 antibody

286 288 290 284

286 288

750 1000

1250

1500

µXPCLµXPCL-PAAcµXPCL-CD34µXPCL-PAAc-NHS/EDC

Wavelength

(a)

5.3.4.2 AFM imaging

AFM imaging shows fibrillar morphology typical of stretched spherulites on PCL films as a result of biaxial stretching (Figure 5-10 a, b) On PCL-PAAc, the fibrils were partially obscured by the engrafted layer, resulting in a drop in roughness (3.2±1.1 nm vs 6.7±1.4 nm, p<0.001) (Figure 5-10c) PCL-CD34 showed a distinct topography in Figure 5-10d where the conjugated CD 34 antibodies were found to adopt a globular morphology Roughness measurements show that PCL-CD34 is smoother than PCL (4.5±0.6 nm, p<0.01), and similar to PCL-PAAc where the difference was insignificant (p>0.05)

PCL PCL-PAAc PCL-CD34 Roughness 6.74±1.43 nm 3.24 ± 1.09 nm 4.46 ± 0.58 nm

Figure 5-10: AFM analysis of modified film surfaces

(a) Three dimensionally rendered images (Scan area 5 µm x 5 µm) of the film surfaces following surfaces PCL film surfaces demonstrate aligned fibres arising from the

biaxial stretching process, which are covered by the grafted polyacrylic acid layer and the conjugated CD34 antibody (b) Similarly, images of the film surfaces captured

using amplitude signal (Scan area 1 µm x 1 µm) shows that the surface is covered by PAAc Conjugated CD34 antibodies show up as globular structures Surface

roughness was evaluated and presented in (c)

5.3.4.3 Carboxyl density

Following engraftment of PAAc to PCL, carboxyl density increased from 2.6±1.0 nmol/cm2 to 170.8±4.4 nmol/cm2 on TBO assay The addition of CD34 antibody conjugation to raised the carboxyl density further to 216.7±7.9 nmol/cm2 (p<0.001)

TBO Assay

0 50 100 150 200 250

Figure 5-11: Surface carboxyl density of surface modified films

Surface carboxyl density of pristine and modified PCL films as assessed by modified Toluidine BlueO assay *** denotes significantly increased carboxyl density over pristine PCL films (p<0.001)

5.3.4.4 Stability

To visualise the distribution of the antibodies, I imaged the films using immunohistochemical techniques The immobilised CD34 antibodies picked up the label, staining the films green In contrast, PCL-PAAc films showed little staining after the antibodies were washed off Subjecting PCL-CD34 to washing under physiological conditions did not deplete the antibodies on the surface, as evidenced by

a retention of fluorescence intensity following 72 hours

Figure 5-12: Fluorescent imaging of PCL-CD34 films

(a) When films were immunostained with fluorescent labels, control PCL-PAAc film did not exhibit fluorescence, as compared to (b) µXPCL-CD34, showing uniform immobilisation of bound antibodies on the film (c) Films were washed under mechanical shaking, and measurements of samples retrieved after 24 and 72 hours Residual Mean Fluoresence Intensity on the films was retained, showing antibodies remain stably bound NS denotes no significant difference (p>0.05)

Mean Fluoresence Intensity

0 10 20 30 40 50

5.4 Discussion

5.4.1 Summary Of Results

µXPCL surfaces are inherently hydrophobic and bioinert, and surface modification is necessary to improve the biocompatibility of the material Wet chemical methods are inappropriate, due to the penetrative nature of such treatments and the microthin nature of the films In particular, bioresorbable polyesters, such as PCL, are highly susceptible to chemical and thermal degradation, and consequently, mechanical properties are often compromised as a result of the modification process Furthermore, large amounts of chemicals are typically involved, which are less environmentally friendly, and potentially toxic if not removed properly (Abidi 2004) Thus, I applied plasma-based techniques to immobilise hydrogel layers on the µXPCL film surfaces, and demonstrated that the mechanical properties were not significantly affected as a result of surface modification In particular, polyacrylic acid could be immobilised on the surface, thus achieving functionalisation of the surface with carboxyl groups, with corresponding reduction in static water contact angle The significant loading of functional groups indicates that the modified surfaces could be used for subsequent conjugation of biomolecules Furthermore, the process could be adjusted to get a range of carboxyl densities, and corresponding range of hydrophilicity for specific applications

after thorough repeated washing My results confirm the feasibility of the use of plasma based methods for the surface modification of µXPCL films without affecting its tensile strength

5.4.2 Critical Assessment

5.4.2.1 Plasma immobilisation of hydrogels

Similar plasma techniques have previously been used to deposit PAAc layers onto bioresorbable nanofiber mats (Park 2007) However, such deposited layers require mechanical interlocking with asperities on the substrate surface, and consequently, are unstable when deposited on smooth substrates (Kato 2003) Although the plasma immobilisation method described here has previously been shown to be inefficient

due to etching effects (Terlingen 1993; Terlingen 1994) Nitschke et al achieved

success with the plasma immobilisation of various hydrogels onto both PET and PE surfaces, suggesting a dependence of grafting efficiency on the following parameters: (1) wettability of substrate (2) thickness of hydrogel layer and (3) molecular weight of PAAc

Using the Nitschke method, I found that PAAc could similarly be engrafted onto the µXPCL surface, and that it was possible to increase the grafting yield simply by repeating the process The plasma immobilisation occurs via radical mechanism (Lens 1998) which first involves the generation of free radicals via chain scission and hydrogen abstraction, followed by crosslinking of the adsorbed polymeric layer This allows the subsequent immobilisation of additional PAAc layers by attachment to underlying layers The process, however, is limited by etching (Terlingen 1994),

resulting in maximum carboxyl density achieved after three repeats of the process Using this method, I achieved a range of carboxyl densities from 100 to 200 nmol/cm2 This figure compares favourably against other groups using other methods, and shown

to be suitable for the subsequent conjugation of biomolecules (Sano 1993; Grondahl 2005; Du 2006)

5.4.2.2 Conjugation of biomolecules

In my experiments with heparin and collagen-conjugated films, I often found the presence of contaminants A likely cause for the contaminations is the source Currently, many biomolecules are derived from animal tissue, and despite stringent quality control, valid concerns have been raised on the quality, purity and predictability of such products Recombinant proteins are increasingly being studied, providing a possible solution to these concerns in the future (Ito 2006) Thus, I have concentrated my investigations on the purer and better characterised monoclonal antibodies, CD34 in my case, synthesised through hybridomas, which should have more predictable results

To promote the adhesion of endothelial progenitor cells (EPC) on µXPCL-PAAc, in order to generate a confluent endothelium in vivo, I demonstrated the use of EPC-selective CD34 antibodies immobilised on the substrate surface to anchor EC types

endothelium, but are lost in terminally differentiated cells Capture of CD34 positive cells may thus improve the healing and tissue integration process Although ubiquitous extracellular matrix proteins such as fibronectin or vitronectin are more commonly used in tissue engineering, such molecules are known to lead to undesirable biological responses such as thrombosis (Stephan 2006)

In this part of the study, however, I have not studied the activity of the immobilised molecule, nor did I manage to quantify the surface density of CD34 antibodies Carbodiimide induced cross-linking has been associated with loss of protein activity, due to the non-specific nature of the process This leads to excessive cross-linking, resulting in changes in protein conformation and loss of activity (Camarero 2008) Furthermore, the cross-links may occur at or near the active site, obscuring the ability

of the antibody to identify the cell antigen In my experiments, I had used titration methods to assess the minimum amount of carbodiimide required to facilitate conjugation, in order to reduce the frequency of cross-linkages However, there are no assays available to study the biological activity of the surface, and I proceeded to characterise the biological response to the modified surface instead The use of recombinant affinity tags and site specific covalent immobilisation have recently emerged as sophisticated methods to address this issue (Holt 2000; Olsson 2000), and may be worth pursuing in the future Another key limitation in the use of carbodiimides and other zero-length cross linkers is steric hindrance This reduces mobility of the bound protein, and may disrupt biological interactions If necessary, the use of linkers and spacers, such as polyethylene glycol, may be used in the future (Kuhl 1996)

6 Surface Modification to Improve Biocompatibility

6.1 Introduction

Common host responses that tissue engineers try to modulate include inflammation, healing and cell adhesion I have described in Chapter 3 the development of microthin, bioresorbable films with slow degradation kinetics to reduce inflammatory responses Cell seeding is commonly employed to accelerate the healing process, and I have detailed in Chapter 4 my studies on candidate vascular progenitor cells I had also described in Chapter 5 modification methods to engineer the film surfaces which can

be conferred with biological functionality through conjugation of biomolecules These developments contribute towards improved biocompatibility of the tissue engineering scaffolds

In the specific context of vascular tissue, two other important classes of tissue responses remain First, possible issues of thrombogenesis must be addressed Acute failures of vascular graft occurring within the first three months of implantation are caused by technical complications, primarily thrombosis The graft is in constant contact with blood, and must perform the homeostatic regulation of blood clot formation Next, vascular grafts typically fail in the mid-term because of re-occlusion arising from neointimal hyperplasia Existing vascular grafts are stiff, resulting in the mechanical mismatch that is believed to direct smooth muscle cells from the media towards a hyperproliferative synthetic phenotype

I have described in Chapter 1 layer-by-layer approach that mimic vascular architecture I describe in this chapter the process of film modification and optimisation for improved biocompatibility Because vascular tissue is organised into

specific to the tunicae Thus, I studied the adhesion of perivascular cells to modified PCL film surfaces for the generation of the perivascular compartment I then proceeded to engineer a surface for the reconstitution of the tunica intima The intima

is constantly exposed to fluid shear stresses, and consequently, suitable methods to anchor the cultured EC are necessary Additionally, the surface may be denuded as a result of the surgical procedure or natural sloughing in vivo, thus the underlying material must be designed to be blood compatible These factor led to my selection of CD34 antibodies as a suitable conjugand, and I detail here my studies on cell adhesion,

as well as blood compatibility

Finally, I studied the assembly of cells and film to form a layered structure The tunicae in vascular tissue are unique in the composition of cells: each compartment is only populated by the resident cell type Thus, endothelial and smooth muscle cells reside as distinct populations in the tunica intima and tunica media respectively, separated by a basement membrane It has been postulated that disruptions of this configuration contribute towards changes in cell phenotype; in particular, disrupted endothelium contributes towards transformation of smooth muscle cells in the media Consequently, it is imperative that the different cell types are maintained distinct from each other prior to the formation of confluent layers I describe here my co-culture studies towards the generation of distinct layers, reminiscent of the native vessel

6.2 Biological Responses To Surface Modified µ XPCL Films

6.2.1 Blood Compatibility Experiments

I chose three aspects of blood clotting to evaluate: Contact activation, platelet activation, as well as whole blood responses

6.2.1.1 Contact activation

TEG profiles generated for each group was found to have classical cigar shapes indicating functional clotting typical of normal haemostasis (Figure 6-1) There were no significant differences in A° and MA across the groups (p>0.05) The TEG profile for PCL was found to

be closest to that of the negative control, with comparable r and MA values The k time, however, was found to be significantly reduced (12.7 min vs 7.5 min, p<0.05) Following PAAc engraftment, contact activation was significantly increased compared to PCL, with significantly reduced r (11.6 min, p<0.001) and k times (3.5 min, p<0.05), but no significant difference in MA There was a trend towards an increased A° in PCL-PAAc over PCL films, albeit insignificantly Subsequent conjugation of CD34 antibody increased both r and k times (19.3 min and 6.9 min respectively, p<0.001) and reduced A° to levels comparable with PCL

Figure 6-1: TEG profiles of blood in contact with modified film surfaces

Glass was included as a positive control and citrated blood was used as a negative control (a) Profiles demonstrate similar cigar shape, indicating functional haemostasis Tracings taken from left to right: Glass, PCL-PAAc, PCL, PCL-CD34 and Negative Control

TEG - Angle

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6.2.1.2 Platelet Adhesion

Widespread and uniform platelet attachment could be found on pristine PCL surface, with 2.5 times as many platelets attaching to PCL than glass (p<0.001) (5,650 vs 2,167, Figure 6-3) PAAc engraftment resulted in a 9-fold decrease in number of adhered platelets compared to PCL (620, p<0.001) The addition of CD34 antibodies resulted in a doubling of attached platelets, although this difference was statistically insignificant (1,389, p>0.05) (Figure 6-3)

Platelet morphology was used as an indication of extent of activation in response to the samples surfaces (Figure 6-4) Adhered platelets found on the pristine PCL films had developed pseudopodia-like structures from the platelet body, with spread hyaloplasm and well-established fibrin networks branching out to the surrounding area, (yellow arrows, Figure 6-4b), similar to those observed on glass (Figure 6-4a)

In contrast, platelets adhering to PCL-PAAc (Figure 6-4c) appeared to be dendritic without evidence of flattening Similarly on PCL-CD34 (Figure 6-4d), platelets were largely dendritic, with some intermediate pseudopodia

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Glass PCL PCL-PAAc PCL-CD34

Figure 6-4: Platelet activation studies

Scanning electron microscope images of adhered platelets on the different surfaces (a) Glass (b) PCL (c) PCL-PAAc and (d) PCL-CD34, displaying distinct morphology (2,000x magnification) In particular, platelets adhering to PCL surfaces appear to have laid down fibrillar fibrin networks (indicated by yellow arrowheads) Scale bar represents 2µm Table represents qualitative assessment of platelet activation status, from 1 (unactivated) to 5 (highly activated)

6.2.1.3 Blood Compatibility Index

The BCI assay was used as a method to evaluate whole blood responses to the material surfaces PCL-PAAc was found to be more thrombogenic than PCL (43.4±2.4% vs 60.9±2.5%, p<0.001) CD34 conjugation was found to significantly improve blood compatibility over both PCL and PCL-PAAc (69.3±3.2%, p<0.001) (Figure 7)

Figure 6-5: Blood Compatibility Index (BCI) Assay

Whole blood response to film surfaces demonstrates that PAAc engraftment renders PCL films thrombogenic This is reversed by conjugation of CD34 antibody, and results in improved blood compatibility over native PCL (* denotes p<0.05)

6.2.2 Cellular Responses To Modified Surfaces

In order to test the cytocompatibility of the modified films, I proceeded to culture vascular progenitor cells on the modified surfaces Conflicting data on the compatibility of smooth muscle cell types with PAAc modified surfaces have previously been described (Bisson 2002; Gupta 2002) Thus, I first studied the influence of PAAc engraftment on UCPVCs

6.2.2.1 Culture of umbilical cord perivascular cells

UCPVC attached, albeit poorly on pristine µXPCL surfaces In contrast, UCPVC seeded on PCL-PAAc attached well, forming more adhesion points with the culture surface and developing a more mature cytoskeleton pattern, more akin to glass (Figure 6-6) Cytocompatibility, as measured by Fluorescein Diacetate / Propidium Iodide (FDA / PI) viability assay was demonstrated, with the majority of cells remaining viable after 7 days of culture in all groups (Figure 6-7) UCPVC were able

to attach well to the PCL-PAAc and thus, rapidly proliferated to form stable multiple cell layers, similar to that found on glass surface

6.2.2.2 Culture of umbilical cord blood endothelial progenitor cells

Subsequently, I cultured umbilical cord blood endothelial progenitor cells (EPC) were cultured on the surfaces In contrast to UCPVC, the seeding of EPC on both µXPCL and µXPCL-PAAc films resulted in poor cellular adhesion (Figure 6-8) and limited

cytoskeletal organisation at day 1 (Day 1, Figure 6-9), with limited proliferation by

day 7 Addition of CD34 antibodies to PCL-PAAc allowed improved cellular

adhesion and proliferation over PCL-PAAc, with cells achieving a more mature cytoskeletal conformation

Figure 6-6: UCPVC adhesion study

UCPVC culture on modified films Cytoskeletal actin was stained using phalloidin (red, counterstained with DAPI; blue) to visualise attached cell structure

Figure 6-7 UCPVC viability study

Cellular proliferation studies with Fluorescein Diacetate (Green) / Propidium Iodide (Red) staining of UCPVC grown on PCL, PCL-PAAc and glass showing improved cellular growth on PCL-PAAc compared to unmodified PCL films after seven days in culture

Figure 6-8: EPC adhesion study

Confocal microscopy of EPC stained with Phalloidin, demonstrating improved development of cytoskeletal structures when grown on PCL-CD34 then on PCL and

PCL-PAAc

Figure 6-9 EPC viability study

Cellular proliferation improved with the surface modification with PAAC, and further

increased with PCL-CD34 films (control – glass) through staining with Fluorescein

Diacetate (Green) / Propidium Iodide (Red) over seven days in culture Tight colonies

were formed on µXPCL-CD34 films after seven days

6.2.2.3 Culture of umbilical cord blood endothelial progenitor cells

By culturing UCPVC and EPC on the opposing sides of µXPCL films modified by

PAAc and CD34 respectively, I observed confluent monolayers for both cell types on

both sides of the modified film over seven days of culture (Figure 6-10) These compartmentalised monolayers retained the expression of smooth muscle actin in UCPVC (Figure 6-10a,b) and VE-Cadherin in EPC (Figure 6-10c,d), and cell types were maintained without cross-contaminations across the film It was also observed that VE-Cadherin expression was lost when serum-containing medium was used

Figure 6-10 Biphasic culture system to recapitulate vascular architecture EPC

(a,b) and UCPVC (c,d) cultured in the intimal and medial compartments respectively Cell populations were maintained distinctively of each other Immunocytochemical evaluation of EPC for VE-Cadherin (red) at 40x magnification (a) and 60x magnification (b) and UCPVC for smooth muscle actin (red) at 40x magnification (a) and 60x magnification (b) to demonstrate retention of cellular phenotype Cell nuclei

Intimal compartment

Medial compartment

(d) (c)

6.3 Discussion

6.3.1 Summary of Results

I have demonstrated in the chapter the enhancement of biocompatibility of µXPCL films for vascular tissue engineering In the context of the application, I have defined blood compatibility and cytocompatibility with vascular cell types to be the most important factors I assessed the blood compatibility of modified and pristine µXPCL films, using anticoagulated blood for negative control, and glass, a known thrombogen, as the positive control Pristine µXPCL was found not to significantly induce contact activation, but demonstrated high platelet adhesion Both trends were reversed by the immobilisation of PAAc The subsequent conjugation of CD34 antibody reduced contact activation, and thus suggesting that blood compatibility had been enhanced This result was borne out by blood compatibility index assay, showing PCL-CD34 to have superior blood compatibility

I then proceeded to study the cellular response to the surface I identified that PAAc is suitable for perivascular cells, and PCL-CD34 to be suitable for endothelial progenitor cell attachment and proliferation Subsequently, I placed them into a layered co-culture system, and demonstrated that the cell layers could be maintained

PCL-6.3.2 Critical Assessment

6.3.2.1 Blood compatibility

In this chapter, I studied the acute haemocompatibility of polycaprolatone, a bioresorbable polymer currently being studied for use in vascular tissue emgineering (Ma 2005; Serrano 2005; Luong-Van 2006) Despite the increasing popularity, few attempts to study the blood compatibility of the material and derivatives have been described in literature and, to my knowledge, only three previous studies having been conducted Yin et al described significant platelet adhesion on PCL surfaces, which was reduced following modification with CD34 antibody (Yin 2009) Stavridi et al described “good haemocompatibility” of PCL, although it should be noted that the industrial grade PCL they used contained contaminants such as plasticisers (Stavridi 2003) Furthermore, the study lacked adequate controls and benchmarks for inferences towards the actual blood compatibility in vivo In a separate study, Liu et al demonstrated that the blood compatibility of PCL could be improved for blood contacting applications thorugh surface incorporation of chitosan, heparin and polyethylene glycol (PEG), resulting in reduced platelet adhesion and delayed plasma recalcification times (Liu 2008) More recently, Yin et al described significant platelet adhesion on PCL surfaces, which was reduced following modification with CD34 antibody (Yin 2009) In this chapter, I presented further evidence to suggest that surface conjugation of CD34 antibody could appreciably improve the blood compatibility of native PCL

Three aspects of blood compatibility were studied to encompass the various components of blood clotting: Contact activation, platelet interaction and whole blood

contact activation of the clotting cascade TEG has been previously shown to reflect the in vivo performance of a blood contacting material accurately (Lemm 1980) Compared to other contact activation assays, such as partial thromboplastin time (PTT) tests, TEG measures the whole blood response, including the size of the clot,, an indication of consumption of clotting proteins Classical cigar-shaped plots could be generated in response to each group with no enhanced fibrinolysis, indicating clotting kinetics was mainly due to coagulation activation process It was observed that the maximum amplitude was preserved across the samples, suggesting negligible protein consumption by the material surfaces Although no significant difference in the rate of clotting, as defined by A°, was measured, the trend appeared to be inversely related to that of the observed ‘k’ values (i.e A°Glass ≈ A°PCL-PAAc>> A°PCL-

CD34 ≈ A°PCL > A° Negative control)

Based on ‘r’ values, PCL, which is inherently hydrophobic, appears to exert little contact activation, similar to the negative control It is proposed that protein layers are quickly established on hydrophobic surfaces following exposure to blood, resulting in adsorption competition between factor XII and other plasma proteins, and the apparent suppression of factor XII activation (Zhuo 2006) In contrast, contact with PCL-PAAc results in significant reduction of clotting times This may be due to the fact that Factor XII, a crucial factor in the intrinsic contact-activation coagulation

5.3.4.3) Thus, when PCL-PAAc is in contact with plasma, deprotonation occurs in the neutral-basic environment (pH 7.2 – 7.6), resulting in a large surface density of anionic hydrophilic carboxylates, which catalyses activation of factor XII

CD34 antibody conjugation was found to reduce the extent of contact activation, despite an almost 30% increase in carboxyl density AFM imaging shows that the PCL-CD34 surfaces are significantly altered due to the presence of conjugated CD34 antibodies, which adopt a three-dimensional globular configuration It follows that the carboxyl groups presented on lateral surfaces of the CD34 antibodies may be accessible to the small TBO molecules, but not to Factor XII molecules, which are

250 times larger Furthermore, protein coatings have previously been employed to create non-thrombogenic surfaces For example, albumin, which lacks cell-binding domains (Poot 1988), is used to passivate surfaces, where it has been shown to demonstrate steric repulsion of platelets and clotting factors Similarly, CD34 antibodies, which specifically promote the adhesion of EPC, will reduce nonspecific adsorption of proteins In contrast to albumin coatings, which repel all proteins and cells indiscriminately, CD34 antibodies anchor EPC to the surface and facilitate the formation of a stable endothelium (Aoki 2005; Chong 2009) Thus, whereas albumin coatings fail over the course of time, due to eventual degradation and displacement by thrombogenic proteins (Eberhart 1987), the EPC capture surface leads to the formation of a stable intima capable of maintaining long term haemocompatibility

Although my results indicate that contact activation by PCL is low, it was accompanied with an unexpectedly high level of platelet adhesion and activation Morphological examination of the attached platelets on PCL showed lamelipodia-like

structures, indicative of extensive, stable interaction and advanced activation (Nachmias 1980), surpassing even that of glass Platelet activation progresses by exocytosis of dense granules, followed by a signalling cascade resulting in a change into an amorphous, spherical form with projecting pseudopodia and finally hyaloplasm to adopt the flattened morphology seen on PCL This heightened effect of PCL on platelet adhesion and activation could be completely abrogated through the addition of polyacrylic acid to the surface While the role of material surfaces on platelet adhesion and activation is still unclear (Ratner 1993), it is likely that these interactions are influenced by the nature of adsorbed protein layers, which in turn, is governed by surface energetics (Vogler 1998) Thus, I suggest that adsorbed layer on the highly hydrophobic PCL film surface may contribute to the poor platelet compatibility of the material In contrast, the increased hydrophilicity and reduction

of water contact angle following PAAc engraftment (See Section 5.2.1.2) exerts both steric and electrostatic repulsion to protein and platelets (Amiji 1993; Lee 2002) Previous studies similarly describe increased contact activation and reduced platelet adhesion to carboxyl groups, as compared to uncharged, hydrophobic methyl groups (Sperling 2005) Again, similar to albumin-conjugated surfaces, conjugated CD34 antibodies serve to sterically repel platelets from the material surface, due to lack of platelet-recognisable domains as well as the presence of a three-dimensional globular configuration, contributing to the observed low levels of platelet adhesion and

Finally, whole blood responses to the various materials were studied using the Blood Compatibility Index assay Despite the obvious differences in platelet responses, the BCI results confirms that PCL is rendered thrombogenic by the PAAc engraftment, and that subsequent CD34 antibody conjugation attenuates this thrombogenic response Furthermore, significant improvement was observed in PCL-CD34 over pristine PCL, providing further support for the use of CD34 antibody conjugation for the improvement of cardiovascular biocompatibility

Thus, I have found PCL-CD34 to have improved compatibility with blood It was not possible, however, to conclude that the material is blood compatible, due to the lack

of an ideal haemocompatible surface to serve as the basis for comparison Nevertheless, the clotting times were found to be insignificantly altered in response with contact with blood, and clotting proteins were not found to be consumed by blood contact with the modified surface

I also recognise that several aspects of haemocompatibility remain to be examined, and a complete assessment would require, but are not limited to, tests to evaluate complement activation, performance under haemodnamic stresses and platelet adhesion in the presence of activating factors, such as von Willebrand factor (vWF),

to give a fair indication of performance in vivo (Sefton 2000) However, the list is by

no means exhaustive, and even extensive testing would not be able to recapitulate the occurrence and interplay of events in vivo Thus, the static assessment was pursued here, with further evaluation pursued using in vivo models