Development of a New Glaucoma Drainage Device

Mr Kin Sheng Lim m b chB FRCOphth

2001

Thesis for the degree o f

Doctor of Medicine (MD)

University o f London

Biomaterials Research GroupDepartment of Pharmacy University of Brighton Cockcroft Building Brighton BN2 4GJ

Wound Healing Research GroupDepartment of Pathology & Glaucoma Institute of Ophthalmology & Moorfields Eye Hospital Bath Street London EClV 9EL

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TABLE OF CONTENTSA B S T R A C TA C K N O W L E D G E M E N T SS T A T E M E N T O F O R G IN A L IT Y A N D P U B L IC A T IO N S A R IS IN G FR O M T H E T H E SIS

CHAPTER ONE - Introduction1.1 Definition o f Glaucoma1.2 Classification1.3 Prevalence o f Glaucoma1.4 Disease Mechanism1.5 Adequate Pressure Reduction1.6 The Need for a New Glaucoma Filtration Procedure1.7 Glaucoma Drainage Devices

1.7.1 Early Devices1.7.2 Contemporary Devices

1.8 Evaluation o f Results1.9 Complication Mechanisms

1.9.1. Poor Flow Control1.9.1.1 Devices with No Resistance Mechanism1.9.1.2 Devices with No Set Resistance1.9.1.3 Devices with Set Resistance

1.9.2 Poor Tissue Compatibility1.10 Strategies to Improve Success Rates

1.10.1 Plate Surface Area1.10.2 Antimetabolites

1.11 Current Indications1.12 New Design Concepts

1.12.1 Tube without Plate1.12.2 New Biomaterial

1.13 The Future

CHAPTER TW O - Design Changes to Control FlowAbstract2.1 Introduction2.2 Fluid Mechanics2.3 Materials & Methods (Internal Flow Control)

2.3.1 Preparation o f Polyimide discs & Excimer Laser Ablations2.3.2 Flow Rig2.3.3 Scanning Electron Microscopy & Image Analysis

2.4 Materials & Methods (External Flow Control)2.4.1 Artificial Anterior Chamber2.4.2 Occluded Device Implantation

2.5 Results2.6 Discussions2.7 Conclusions

CHAPTER THREE - Biocompatibility StudiesAbstract3.1 Introduction3.2 Bioinert Materials

3.2.1 Heparin Coating3.2.2 Surface Passivation3.2.3 Hydrogels3.2.4 Polyethylene Oxide Polymers3.2.5 Biomimetic Bioinert Materials

3.3 Biocompatibility in the Context o f Glaucoma Drainage Devices3.3.1 Protein & Cell Adhesion - Tube Occlusion3.3.2 Protein & Cell Adhesion - Chronic Inflammation & Bleb

Fibrosis3.3.3 Cell Damage - Endothelial Failure3.3.4 Bacteria Adhesion - Endophthalmitis

3.4 Aims3.5 Materials & Methods

3.5.1 Preparation o f Discs3.5.2 Cell & Protein Adhesion

3.5.2.1 Fibrinogen Adsorption3.5.2.2 Fibrin Adhesion3.5.2.3 Human Scleral Fibroblast Adhesion3.5.2.4 Macrophage Adhesion3.5.2.5 Data Analysis

3.5.3 Endothelial Damage3.5.4 Bacteria Adhesion

3.6 Results3.6.1 Protein & Cell Adhesion3.6.2 Endothelial Damage3.6.3 Bacteria Adhesion

3.7 Discussion3.7.1 Choice o f Biomaterials3.7.2 Cell & Protein Adhesion3.7.3 Endothelial Damage3.7.4 Bacteria Adhesion

3.8 Conelusion

CHAPTER FOUR - Prototyping & In Vivo StudyAbstract4.1 Introduction4.2 Materials & Methods

4.2.1 Prototyping4.2.2 Flow Rig4.2.3 Implantation o f Prototype Glaucoma Drainage Device4.2.4 Follow-up Examination4.2.5 Sacrifice

4.3 Results4.3.1 Pre-operative Flow Testing4.3.2 Adverse Reactions4.3.3 Intraocular Pressure

4.4 Discussion4.5 Conclusion

CHAPTER FIVE - Concluding Discussion & Future Development5.1 Discussion5.2 Future Development

5.2.1 Extrusion Moulding & Hot Drawing5.2.2 Photolithography

5.3 Conclusion

REFERENCES

APPENDICES

ABSTRACT

Glaucoma filtration surgery has been shown to be more effective at preventing disease

progression than other primary treatment modalities in open angle glaucoma. If it

were possible to avoid complications associated with poor flow control, primary

glaucoma filtration surgery would probably be offered more widely. Trabeculectomy,

the procedure o f choice in conventional glaucoma filtration surgery, has remained

essentially unchanged for over a quarter o f a century. Local control over wound

healing with antimetabolite agents such as 5-fluorouracil and mitomycin C has

improved the prognosis for cases with high risk o f filtration failure; but flow control

remains imprecise despite the introduction o f a variety o f suture adjustment

techniques. Glaucoma drainage devices have the potential to regulate flow

consistently, eliminating hypotony after surgery. Design, material, and manufacturing

deficiencies have left this potential unfulfilled in existing glaucoma drainage devices,

all o f which exhibit problems with poor flow control and suboptimal tissue

compatibility.

This thesis will examine whether a new flow resistor produced by excimer laser

ablation in a polymer substrate and a new external glaucoma drainage device design

could prevent hypotony in a series o f in vitro experiments and a limited in vivo study.

It will also investigate whether phosphorylcholine coating o f polymethyl methacrylate

can reduce the adhesion o f cells and proteins compared to current biomaterials used in

the construction o f glaucoma drainage devices.

PUBLICATIONS AND PRESENTATIONS ARISING FROM THE THESIS & STATEMENT OF ORIGINALITY

Publications

Glaucoma filtration implants. Past, present and future.Lim KS, Allan B, Muir A, Lloyd AW, Khaw FT.British Journal o f Ophthalmology 1998;82(9); 1083-1089

Cell and protein adhesion studies in glaucoma drainage device development Lim KS, Allan B, Muir A, Lloyd AW, Khaw FT, et al.British Journal o f Ophthalmology 1999; 83: 1168-1171

Experimental flow studies in the development o f glaucoma drainage device Lim KS, Allan B, Muir A, Lloyd AW, Khaw FT, et al.British Journal o f Ophthalmology (in press)

Glaucoma valves: Truth versus myth II Lim KS, Allan B, Khaw FT.Ophthalmology (in press)

Comeal endothelial cell damage fi"om glaucoma drainage devices’ materials Lim KS, Allan B, Muir A, Lloyd AW, Khaw FT, et al.Submitted to Cornea

Presentations

Biomimetic coatings to reduce fibrin blockage o f glaucoma filtration implants.Allan B, Lim KS, Dropcova S, Muir A, Lloyd AW, Khaw FT, et al. 1997 Investigative Ophthalmology & Visual Science', 38(4):1252-1252

Consistent Flow Control in a New Glaucoma Filtration Implant.Lim KS, Allan B, Muir A, Lloyd AW, Khaw FT, et al. 1998 Investigative Ophthalmology & Visual Science', 39(4):2176-S475

In Vitro Ocular Compatibility o f Glaucoma Filtration Implant Materials.Denyer SF, Lim KS, Faragher RGA, Allan B, Muir A, Lloyd AW, Khaw FT, et al. 1998 Investigative Ophthalmology & Visual Science', 39(4):2176-S475

Comeal Endothelial Cell DamageLim KS, Allan B, Muir A, Lloyd AW, Khaw FT, et al.Investigative Ophthalmology & Visual Science', 1999

STATEMENT OF ORIGINALITY

The experiments were carried out personally. The experimental designs were conceived and designed by myself with guidance firom my supervisors.

ACK NO W LEDG EM ENTS

This thesis simply would not exist without Bruce Allan. I would like to thank Bruce for his tireless support throughout the project and also the countless hours spent correcting this thesis.

I am extremely thankful to Peng Khaw and Andrew Lloyd for their support and advice, Richard Faragher, for his help with the biocompatibility studies, and everyone from the Biomaterial Research and Wound Healing Research Groups for all their help and friendship.

Finally, I would like to express my sincere gratitude to the Department o f Trade and Industry, the Department o f Health and International Glaucoma Association for their financial support thus made all the work possible.

To my paren ts

CHAPTER ONE - Introduction

1.1 Definition of Glaucoma

The glaucomas are a group o f disorders characterised by optic nerve head damage,

visual field loss and an intraocular pressure (IGF) sufficiently raised to affect the

functioning o f the optic nerve head.

1.2 Classification

Glaucomas are most commonly classified as either open or closed angle, and may

occur in isolation (primary glaucomas) or in association with other ocular pathology

(secondary glaucomas). In open angle glaucoma, the primary cause o f raised 10? is

increased resistance to fluid drainage within the trabecular mesh work. In closed angle

glaucomas, iris occlusion o f the anterior chamber drainage angle prevents aqueous

escape.

1.3 Prevalence o f Glaucoma

Glaucoma is a leading worldwide cause o f blindness. Pooled estimates from a variety

o f population studies suggest an overall prevalence o f around 0.5% for primary open

angle glaucoma, rising with age to 2% in those aged 70-75 years (Hart 1989).

Analysis by Quigley (Quigley 1996), using population projections, suggested that

there would be around 66.7 million people with primary glaucoma worldwide in the

year 2000, with 6.7 million suffering from bilateral blindness. In England, glaucoma

10

is the second commonest cause o f severe visual impairment, accounting for

approximately 15% o f blind registrations (Grey 1989).

1.4 Disease Mechanism

Although the pathogenesis o f glaucoma is not fully understood, the disease

mechanism proposed by Maumenee (Maumenee 1983) is widely accepted. Raised

lOP leads to outward bowing o f the ocular coats at their weakest point: the lamina

cribosa. Increasing obliquity and distortion o f pores in the lamina cribosa strangles

optic nerve fibres as they exit, interrupting axoplasmic flow. Retinal ganglion cell

death follows. With progressive posterior bowing o f the lamina cribosa and nerve

fibre attrition, the optic disc becomes cupped in appearance, and visual field defects

advance. Non-IOP mediated vascular mechanisms o f glaucomatous damage may exist

(Schulzer 1990), but their identification remains incomplete. Direct evidence that an

adequate reduction in lOP will arrest disease progression is provided by studies

involving normal tension glaucoma (Collarborative NTG study group 1998; Membrey

2000). Although results from primary open angle glaucoma patients are not yet

available, they are widely expected to reaffirm this finding.

1.5 Adequate Pressure Reduction

The widely quoted figure o f 21mmHg for the upper limit o f normal lOP is derived

from population studies showing a Gaussian lOP distribution with 20.5mmHg at two

standard deviations above the mean (Schottenstein 1989). This figure has little

functional significance, since progression o f glaucomatous visual field loss can

11

certainly occur at a lower pressure level (Drance 1992). Hence studies demonstrating

continued visual field loss despite normalisation o f the lOP after filtration surgery

may simply demonstrate pressure control at a statistically normal, but functionally

inadequate level (Table 1). Currently, it is generally accepted that the greater the

existing damage to the optic nerve head, the lower the TOP needs to be to prevent

further functional damage (Grant & Burke 1982; Jay & Allan 1989). Mao et al (1991)

reviewed data from a group o f 55 glaucomatous patients with early disc damage

followed for 4-11 years. One eye was selected randomly for study in each patient.

They found no disease progression in patients with a mean lOP level <17 mmHg, and

universal progression in patients with a mean TOP level > 21 mmHg. lOP control at a

lower level (<15 mmHg) may be required where disc damage is more advanced

(Grant & Burke 1982) or in progressive normal pressure glaucoma (Hitchings 1992).

Unfortunately, no methods are available to determine, for any given optic nerve, the

pressure level required to prevent the onset or progression o f damage (Drance 1992).

Even if this guidance were available, information about lOP control gained by

snapshot readings at intermittent clinic visits is necessarily incomplete. But decisions

in medicine are rarely taken on a basis o f absolute certainty. In broad terms, it is

reasonable to conclude that simply reducing the lOP to a statistically normal level

(<21 mmHg) will often be inadequate; and that methods producing TOP control in the

low teens (<15 mmHg) are preferable to those in which TOP control in the high teens

(15-21 mmHg) is a more usual result (Table 1 ).

12

Table 1 Final intraocular pressure after glaucoma surgery and visual prognosis. Pooled analysis from various studies, (adapted from Khaw et al 1995)

Mean lO P after surgery Worse Follow-up Authors15.0 mmHg 18% 5 years Kidd, O ’Connor 198515.7 mmHg 10% 4+ years Kolker 197716.0 mmHg 35% 3.5 years Werner et al 197717.3 mmHg 35% 4 years Greve, Dake 197918.1 mmHg 29% 5 years Rollins, Drance 198119.1 mmHg 58% 5 years Roth et al 1988

1.6 The need for a new glaucoma filtration procedure

Current conventional practice is to use medical therapy, aimed at lowering lOP, as the

primary treatment modality in primary open-angle glaucoma. Argon laser

trabeculoplasty (ALT) is occasionally offered when medical therapy fails to provide

satisfactory lOP control. Glaucoma filtration surgery (GPS) is usually only offered to

patients who have failed either or both o f the other treatment modalities.

Randomised trials comparing these modalities in the initial therapy o f primary open-

angle glaucoma, show trabeculectomy to be more effective than medical treatments or

ALT (Jay & Allan 1992; Migdal 1992) in preventing progressive visual field loss.

These trials also demonstrate lOP control at a lower average level for GPS in

comparison with drug treatments (Jay 1992; Migdal 1992). In addition, prolonged use

o f topical anti-glaucoma medications as a primary treatment modality can increase the

probability o f drainage failure in subsequent GPS (Broadway et al 1994, Lavin et al

1990).

These studies pre-date newer generation topical treatments such as Brimonidine

(Melamed 2000) and Latanoprost (Nordmann 2000), and the use o f antimetabolites in

13

primary trabeculectomy for high risk patients. Whatever the true picture regarding the

relative efficacy o f newer drug treatments and GFS, there are powerful health

economic arguments for preferring a one-stop intervention to lifelong drug therapy.

Drug therapies are also impractical in the developing world. GFS would probably be

offered widely as the primary treatment in glaucoma if techniques were safer and

quicker.

Complications associated with GFS are listed in Table 2. The most important early

postoperative complications in GFS are associated with poor flow control and

resultant hypotony (I0PD5m m Hg). These include anterior chamber flattening, and

choroidal detachment, hypotony maculopathy (Costa et al 1993; Stamper et al 1992;

Cheung et al 1997)) and delayed suprachoroidal haemorrhage (Paysee 1996; Gressel

1984).

Poor initial flow control may also compromise later filtration function. The aqueous

concentration o f fibroblast stimulating cytokines (Joseph et al 1989) is increased in

conditions o f blood aqueous barrier breakdown promoted by hypotony (Addicks et al

1983), and an association between prolonged postoperative hypotony and a higher

final lOP has been observed (Migdal & Hitchings 1988; Blondeau & Phelps 1981).

14

Table 2. Complications associated with trabeculectomy with or without antimetabolites

ComplicationsChoroidal effusionHyphaemaHypotony maculopathyShallow anterior chamberIris to comeal touchSuprachoroidal haemorrhageW ound leakW ound dehiscenceBlebitis/EndophthalmitisCataractFailure

Trabeculectomy, the procedure o f choice in conventional GFS, has remained

essentially unchanged for over a quarter o f a century. Local control over wound

healing with antimetabolite agents such as 5-fluorouracil and mitomycin C has

improved the prognosis for cases with a high risk o f filtration failure (Katz 1995); but

flow control remains inexact despite the introduction o f a variety o f suture adjustment

techniques (Raina 1998, Blok 1992, Singh 1996). Early postoperative hypotony has

been observed in 25-75% o f cases either initially or after suture removal (Blok et al

1992; Fluorouracil filtering surgery study group 1989).

Trabeculectomy is also relatively slow and technically demanding, taking up to two to

three times as long as current cataract operations. A variety o f laser sclerostomy

techniques were developed in an attempt to make GFS simpler and quicker (Allan

1994, Iwach 1996, Wetzel 1994, Latina 1992). These techniques had a universally

poor safety profile, and were not widely practised. Again, the problem was poor flow

control. Attempts to regulate sclerostomy diameter and the resulting outflow

15

resistance were unsuccessful (Allan 1994), and all laser sclerostomy procedures were

effectively unguarded filtration operations, with hypotony in almost every case.

In the last few years, non-penetrating deep sclerectomy has gained popularity as a

safer alternative to trabeculectomy. The main advantage o f this procedure lie in the

fact that the globe is not penetrated during the surgery as a thin layer o f trabecular

meshwork tissue is left (Karlen 1999). This thin layer o f tissue is thought to provide

enough outflow resistance to prevent post-operative hypotony. Further more, as the

globe is not penetrated, no peripheral iridectomy is performed and this results in less

hyphaema and post-operative intraocular inflammation. However, the scope for

human error in regulating factors such as dissection depth in deep sclerectomy, is

considerable, and complication such as inadvertent perforation o f the trabecular

meshwork is common (Karlen 1999). With the limited short term follow up data

available from randomised prospective trials comparing this technique with

trabeculectomy; this new procedure has not established any clear advantage either in

complication or success rate (El Sayyad 2000, Gandolfi 2000).

A new GFS procedure is required which is both simple to perform and offers

consistent protection from over drainage. Flow control after conventional and laser

techniques remains suboptimal. Exquisite dimensional control is required to impart

consistent flow resistance in a fine bore sclerostomy channel. Even if this level o f

control were available, it is likely that dimensions o f cut tissue surfaces would quickly

be modified by biological spoliation. The logical alternative is a glaucoma drainage

device (GDD) in which key dimensions are reproduced in a controlled manufacturing

16

environment, and materials resistant to biological spoliation are used to help ensure

that flow resistance characteristics are preserved after implantation.

1.7 Glaucoma Drainage Devices

Glaucoma drainage devices (GDDs) have the potential to regulate flow consistently,

eliminating hypotony after GFS. Design, material, and manufacturing deficiencies

have left this potential unfulfilled in existing GDDs, all o f which exhibit problems

with poor flow control and suboptimal tissue compatibility. The role o f GDDs in

contemporary GFS remains poorly defined, but possibilities offered by new

biomaterials and the goal o f accurate flow control have stimulated considerable recent

interest in GDD development.

1.7.1 Early Devices

In 1906, horsehair (Rollett 1906) was placed through a comeal paracentesis in an

attempt to drain a hypopyon externally. The same technique was later used to treat

two patients with painful absolute glaucoma (Rollett 1907). Sporadic attempts using

implants to shunt aqueous to a variety o f unconventional sites, including the vortex

veins (Lee 1974), and the nasolacrimal duct (Mascati 1967) have since been reported.

Results were generally unfavourable or too poorly documented to evaluate, and

attention has focused on devices shunting aqueous fluid to the subconjunctival space

as with conventional GFS.

17

The first transi imbal GDD, reported by Zorab in 1912, was silk thread used as a seton

to aid drainage o f anterior chamber fluid to the subconjunctival space. This was

followed by similar use o f gold (Stefansson 1925), tantalum (Bick 1949) and platinum

thread/wire (Muldoon 1951). Results were universally poor however. These and other

early translimbal setons (Table 3) did not address lack o f flow control and hypotony

associated with full thickness (unguarded) GFS, and added a foreign body chronic

inflammatory stimulus. Simple translimbal tube devices (La Rocca 1958, Ellis 1960)

were similarly unsuccessful, with high rates o f early filtration failure.

Translimbal drainage implants, or anterior GDDs, were implanted with the intention

o f preventing filtration failure by maintaining patency o f a drainage fistula or

sclerostomy. Anterior GDDs failed to improve filtration failure rates in comparison

with conventional GFS, but it took almost half a century for investigators to begin to

rationalise this lack o f success.

Table 3. Developments o f glaucoma drainage devices.

18

Year Investigator Type M aterial M ethod Flow Control D rainage Site

1907 Rollet seton Horse hair Paracentesis None External cornea

1912 Zorab seton Silk thread Translimbal None Anteriorsubconjunctival

1925 Stefansson seton/tube Gold Translimbal None Anteriorsubconjunctival

1934 Row seton Platinum Cyclodialysis None Suprachoroidal

1940 Troncoso seton Magnesium Cyclodialysis None Suprachoroidal

1942 Gibson tube Lacrimal canaliculus Transcleral None Anteriorsubconjunctival

1949 Bick seton/tube Tantalum Cyclodialysis None Suprachoroidal

1951 Muldoon seton Platinum Translimbal None Anteriorsubconjunctival

1952 Losche tube Supramid Cyclodialysis None Suprachoroidal

1955 Bietti tube Polyethylene Cyclodialysis None Suprachoroidal

1958 La Rocca tube Polyvinyl Translimbal None Anteriorsubconjunctival

1960 Ellis tube Silicone Translimbal None Anteriorsubconjunctival

1967 Mascati tube Plastic Translimbal None Lacrimal sac

1969 Molteno tube & plate Acrylic Translimbal None Anteriorsubconjunctival

1974 Lee & Wong tube Collagen Translimbal None Vortex vein

1976 Krupin tube Silicone & Supramid Translimbal Slit valve Anteriorsubconjunctival

1979 Honrubia tube Silicone Translimbal None Anteriorsubconjunctival

1982 Schocket tube & band Silicone Translimbal None Posteriorsubconjunctival

1985 White tube & plate Silicone Silicone Valve & pump Posteriorsubconjunctival

1986 Joseph tube & band Silicone Translimbal Slit valve Posteriorsubconjunctival

1990 Krupin tube & plate Silicone Translimbal Slit valve Posteriorsubconjunctival

1990 Baerveldt1991)

tube & plate Silicone Translimbal None Posteriorsubconjunctival

1993 Ahmed1995)

tube & plate Silicone & Polypropylene Translimbal Venturi valve Posteriorsubconjunctival

1995 OptiMed1996)

tube & plate Silicone & PMMA Translimbal Microtubules Posteriorsubconjunctival

1995 Smith seton Hydrogel Translimbal None Intrascleral

1996 Pandya tube & plate Silicone & Hydroxylapatite

Translimbal None Posteriorsubconjunctival

1997 Glovinsky & Belkin

tube Stainless steel Translimbal None Anteriorsubconjunctival

1997 Helies artificialmeshwork

PTFE Transcleral None Anteriorsubconjunctival

19

In 1969, Molteno (Molteno 1969) hypothesised that filtration failure was primarily

attributable to subconjunctival fibrosis, with fistula closure occurring as a secondary

event. This was later confirmed in histological studies o f animal models o f GFS

(Miller et al 1989 and Jampel et al 1990). Realising that simple anterior GDDs would

have little impact on this process, Molteno launched the concept o f tube and plate

GDDs, in which aqueous fluid is shunted to a plate device designed to maintain

patency o f a subconjunctival filtration reservoir in the face o f continuing

subconjunctival fibrosis. Although confined to use in complex cases by the advent o f

trabeculectomy and relatively successful conventional guarded GFS, these were the

first GDDs to gain widespread acceptance and the Molteno tube remains the

benchmark against which other tube devices are compared.

1.7.2 Contemporary Devices

Tube and plate devices dominate the contemporary GDD market. Prominent

examples, in chronological order, are the Molteno, Krupin, Baerveldt, Ahmed, and

OptiMed GDDs (Figures 1-5). Molteno (1976) moved the plate element o f his early

devices posteriorly away from the limbus to avoid problems with dellen formation

and poor filtration associated with pre-existing anterior conjunctival scarring.

Posterior placement beneath Tenon's capsule was also thought to improve protection

from extrusion (Molteno 1976, Melamed 1990). Subsequent tube and plate GDDs

share the essential design concept o f posterior filtration via a tube in the anterior

chamber to a plate element secured beneath Tenon's capsule, but differ in plate design

20

and their provision for a flow control mechanism to protect from early postoperative

hypotony (Table 4).

Table 4. Contemporary Glaucoma Drainage Devices.

G DDs Year o f Introduction

T ube D iam eter/ M aterial

Plate Size/ M aterial

ResistanceM echanism

Molteno 1979 0.63mm OD 0.30mm ID Silicone

135mm^Polypropylene

None

Baerveldt 1990 0.63mm OD 0.30mm ID Silicone

200,250,350.425,500mm"Silicone

None

Krupin with Disk 1990 0.58mm OD 0.38mm ID Silicone

180mm"Silicone

Slit Valve

Ahmed 1993 0.63mm OD 0.30mm ID Silicone

185mm"Polypropylene with Silicone valve

Venturi Valve

OptiMed Model- 1014

1995 0.56mm OD 0.30mm ID Silicone

140 mm"Silicone with PMMA matrix

Microtubules

OD = outside diameter ID = inside diameter

21

Figure la . Single plate M olteno implant. (Scale: bar = 1 cm)

Figure lb. Dual chamber double plate Molteno implant. (Scale: bar = 1 cm)

Figure le. A schematic drawing of the resistance mechanism of a dual chamber double-plate Molteno implant. The thin V-shaped ridge (see Figure lb) has the same height as the circumferential rim of the polypropylene plate. The top surface of the plate is divided into one smaller and one larger chamber by the apposition of the overlying conjunctival and Tenon’s layers (dashed line). Aqueous flows (black arrow) into the smaller proximal chamber until sufficient pressure is achieved within the chamber to lift (white arrow) the overlying conjunctival layer to allow free drainage.

Sclera

22

Figure 2a. The Krupin valve with disk, slit valve is situated at the plate end of the tube. (Scale: bar = 1 cm)

Figure 2b. A schematic drawing of the Krupin slit valve, showing the crossed slit elements.

Direction of Flow

Figure 3a. The Baerveldt implant. (Scale: bar = 1 cm)

23

(■■Ms.

I '

-#:T

Figure 3b. A schematic drawing of the appositional resistance mechanism included in some embodiments of the Baerveldt GDD. An annular ridge projecting from the underside of the plate element provides a temporary seal against the sclera. Absorbable sutures are used to hold the plate in apposition. As the sutures degrade, the plate element lifts clear, allowing free aqueous drainage.

i l l ' V i . '

24

Figure 4a. The Ahm ed glaucoma valve implant. (Scale: bar = 1 cm)

Figure 4b. A schematic drawing of the resistance mechanism of the Ahmed valve. Aqueous flows (black arrow) through the tube into a chamber within the plate element. A folded over silicone membrane (black line) forms this chamber with its free edges forming a one-way valve. The manufacturers claim that the two halves of the polypropylene body of the plate element pre-tension the valve to open at a specific level of lOP. They also claim that the venturi effect produced by the tapering trapezoidal shape of the space enclosed by the folded silicone membrane acts to improve flow regulation (increasing fluid velocity as the chamber tapers acts to reduce internal pressure proximal to the slit opening in accordance with the inverse relationship between fluid velocity and pressure expressed in the Bernoulli’s theorem). Neither of these claims is supported by published experimental evidence.

B

25

Figure 5a. The OptiMed implant is made up of a silicone tube with a PMMA plate. The “flow-restricting” element of this device is housed within the rectangular box situated at the end of the tube within the plate. (Scale: bar = 1 cm)

Figure 5b. A schematic drawing of the OptiMed implant’s “flow-restricting” unit, which is made up of multiple microtubules providing a pressure gradient, governed by Poiseuille’s formula (Appendix 1).

B

Direction of flow

26

1.8 Evaluation of Results

Most GDDs have been developed with little available data to substantiate

manufacturers’ claims for flow performance (Prata et al 1995) or biocompatibility.

Clinical data is largely restricted to uncontrolled retrospective case series (Krawczyk

1995) with variable follow-up and differing definitions o f surgical success. Evaluation

is further complicated by the heterogeneity o f inclusion criteria. Series include a

variable proportion o f complex cases, neovascular glaucoma in particular, with a

predetermined high risk o f filtration failure.

Existing results are summarised in table 3. Overall success rates, in terms o f IGF

control, appear similar between devices (Table 5), with a reasonably high proportion

o f cases achieving a final 10? in the target range at one year after surgery. H alf to two

thirds o f these cases still require glaucoma medications however; and, as already

argued, target lOPs in the low teens (<16mmHg) may be more realistic in terms o f

preventing disease progression than commonly adopted target levels (<21 or

22mmHg).

Another important caveat concerns attrition rates, or continued increments in the

proportion o f filtration failures with lengthening postoperative follow-up. Again,

evaluation is difficult, with few series including either long-term data or survival

analysis. Mills (1996) (Table 5) reported a 10% failure rate per post-operative year in

a series including longer term follow-up for single and double plate Molteno tubes.

Extrapolating from this, it would appear that most GDDs have a functional lifespan o f

less than five years before failure though fibrous encapsulation.

27

Table 5. Success Rates o f current Glaucoma drainage devices.

Type Investigator Year Diagnosis No.ofEyes

Follow-Up (Months) Mean ± SD

Definition of Success*

Success RatewithoutMedication

Success Rate with Medications

M olteno SP & D P M ills 1997 Mixed (25%

Neovascular)77 44/+ lO P < 22 m m H g 23% 3 4 %

SP M erm oud 1993 Neovascular 60 24 .7± 13 .4 I 0 P < 2 1 17% 20%SP

DP

H euer 1992 Mixed (No Neovascular)

50

52

14.9±8.9

16.4 ± 6 .8

5 < I 0 P < 2 1 10%

12%

40%

63%

SP M inckler 1988 Mixed (50% Neovascular)

90 17.6 I 0 P < 2 1 7% 40%

Ahmed C olem an 1995 Mixed 60 9.3++ lO P < 22+ NA NA(78%)^

C olem an 1997 PenetratingKeratoplasty**

31 16++ I0 P < 2 2 + " 26% 39%

C olem an 1997 Paediatric-Mixed

24 16.3±11.2 lO P < 22+ 33% 38%

KrupinDisk

K rupin S tudy G roup

1994 Mixed 50 25 .4± 2 .4 IO P < 19 33% 47%

Fellenbaum 1994 Mixed 25 13.2 6 < 1 0 P < 2 1 28% 36%Baerveldt200 ,250 ,350,500m m "

Siegner 1995 Mixed 103 13.6±0.9 5 < IO P < 2 2 27% 45%

200,350 ,500mm"

Sidoti 1995 Neovascular 36 15.7±7.2 6 < I O P < 2 1 17% 33%

350 mm" Lloyd 1994 Mixed 37 15.5±4.8 6 < 1 0 P < 2 1 14% 70%

500 mm" 36 14.1±5.4 36% 47%

* With no further glaucoma surgery or devastating complication** Without graft failure § Total success rate only §§ Concurrent or Prior+ Or reduction o f >20% if pre-operative IOP>22 mmHg. Data from same patients may appear in more than one o f these three series.++ Median follow-ups in months. Mean values not available in these reports, making direct comparison difficult Molteno SP = Molteno single plate Molteno DP = Molteno double plates

28

1.9 Complication Mechanisms

Clinical series reporting ODD procedures are characterised by frequent problems in

addition to filtration failure (Table 6), with overall complication rates typically

around 60 - 70% (Siegner 1995, Heuer 1992). W hilst partly attributable to the

complex nature o f cases most usually selected for implantation, the range o f

complications observed also reflects design and material inadequacies inherent in

contemporary GDDs.

The origin o f most complications can be traced to just two fundamental mechanisms:

poor flow control and suboptimal material biocompatibility.

Table 6. Cumulative complication rates in the currently used GDDs.

29

Com plications Ahmed Valve Krupin Valve with Disk

BaerveldtIm plant

M oltenoIm plant

(n=115)C olem an 1995 C olem an 1997 C olem an 1997

(n=75)K rupin S tudy G roup 1994Fellenbaum 1994

(n=249)L loyd 1994 S idoti 1995 Sm ith 1993 S iegner 1995

(n=395)M ills 1996 M inck ler 1988 L loyd 1992 H euer 1992

H ypo tony /F la t an te rio r C ham ber

3.5% 27% 16.5% 5%

C horoidalE ffusion /H aem orrhage

14% 33% 23% 7%

Suprachoro idalH aem orrhage

3.5% 1.3% 0.8% 0.5%

H yphaem a 0 1.3% 11% 4%V itreous H aem orrhage 3.5% 1.3% 7% 2%U veitis 1.7% 9.3% 3.6% 0 .5%M alignan t G laucom a 0.9% 2.7% 2% 0.5%M otility Problem 3.5% 4% 21% 0.3%C ataract N /A N /A 9.9% § 12%*T ube B locked 7.8% 11% 10 6%T ube R etraction 5.2% 2.7% 2.4% 0 .8%T ube/P la te E xtrusion 4.3% N /A 1.2 0 .5%T ube/P la te Erosion 0 2.7% 2 3%C orneal Touch 2.6% N /A 4% 3%C orneal D ecom pensation 3.5% 5.3% 11.6% 14%R etinal D etachm ent 0 0 5% 3.5%E ncapsu la ted B leb 3.5% 1.3% 1.6% N /APhthisis 0 1.3% 2.4% 4%O thers 1.7% Wound Leak 1.3% Wound Leak

1.3% Endophthalmitis 1.3% Decompression Retinopathy

1.6% Wound Leak 0.4%Endophthalmitis

0.3% Epiretinal Membrane 0.3% Perforation

* cataract developed in 11 out o f 91 phakic patients. Mean follow-up more than 40 months.§ cataract developed in 7 out o f 69 phakic patients. Mean follow-up 14.4 months.

30

1.9.1 Poor Flow Control

Modifications to GDDs and implantation techniques since Molteno's original device

have largely been driven by attempts to minimise rates o f early postoperative

hypotony. Hypotony is associated with a catalogue o f early and late complications in

GFS, including anterior chamber shallowing, maculopathy, delayed suprachoroidal

haemorrhage and late filtration failure (see 1.6 above). Anterior chamber flattening is

especially hazardous in the context o f GDD implantation in which contact with the

tube element can cause significant comeal endothelial and lens epithelial damage.

GDDs can be categorised into those with no internal resistance mechanism, no set

internal resistance, and those aiming to provide set internal flow resistance.

1.9.1.1 Devices with No Resistance Mechanism

Early Molteno and Baerveldt implants were simple tube and plate devices with no

internal resistance mechanism.

After GFS, resistance to flow distal to the sclerostomy or GDD generally remains low

until limited subconjunctival wound healing has occurred and the initially diffuse

aqueous escape becomes confined within a maturing filtration bleb. Having observed

frequent problems with hypotony in early single stage implantation (Molteno 1977),

Molteno recognised that some allowance for this early period o f minimal distal flow

resistance would be required. A two-stage procedure was initially explored (Molteno

31

1983), in which the device was implanted and allowed to encapsulate prior to

insertion o f the tube element into the anterior chamber at a second operation after 2-6

weeks. In addition to a second operation, a separate provision for initial lOP control in

the intervening period was required, and while the two stage approach was successful

in reducing problems with hypotony, variations o f a modified single stage procedure

are now widely preferred for GDDs with no internal resistance mechanism. These

include ligation with an absorbable suture (Molteno 1986), laser suture-lysis (Price

1989), and occlusion with a supramid stent which is later removed through a small

conjunctival incision (Egbert 1983). Single stage methods seek to occlude the lumen

o f the tube element temporarily to allow partial encapsulation o f the plate. They rely

on external leakage with or without a slit incision through the subconjunctival portion

o f the tube proximal to the occlusion to provide initial outflow. Neither o f these initial

flow mechanisms is well controlled, and problems with either too much or too little

initial aqueous escape remain frequent.

1.9.1.2 Devices with No Set Resistance

Later incarnations o f the Molteno and Baerveldt GDD incorporate resistance

mechanisms, which depend on tissue apposition to limit flow.

The Molteno dual ridge device seeks to limit the initial drainage area by dividing the

top part o f the plate into two separate spaces (Figure lb & 1C). Aqueous escapes

directly into the channel between two concentric ridges on the plate element, but must

overcome resistance associated with conjunctival tissue apposition to flow further.

With later partial encapsulation o f the plate element, the overlying tissues balloon

32

clear o f the inner pressure ridge, and aqueous flow into the space overlying the plate

is unrestricted (Freeman et al 1992).

Apposition o f the "Bioseal" element o f the modified Baerveldt implant to the sclera

with absorbable sutures (Figure 3b) also aims to provide early flow resistance,

limiting initial aqueous escape from beneath the device (Baerveldt 1997).

The essential problem with both approaches is that, as with trabeculectomy, the force

o f tissue apposition is poorly controlled. Early flow resistance varies and initial lOP

levels remain unpredictable.

1.9.1.3 Devices with Set Resistance

Devices which aim to set the initial lOP level by incorporating a non-adjustable

resistance mechanism include the Krupin (Figure 2b), Ahmed (Figure 4b), and

OpitMed (Appendix 1) (Figure 5b) GDDs.

Early independent examination o f the flow characteristics for each o f these deviees

suggests a wide divergence between observed function and manufacturers claims for

flow resistance (Prata et al 1995). Valved devices (Ahmed and Krupin) appear not to

close after initial opening in perfusion tests at physiological flow rates. Resistance

values also vary considerably between devices from the same manufaeturer,

indicating deficiencies in quality control (Porter et al 1997). Although subsequent

flow examinations (Eisenberg 1999, Francis 1998) showed that Ahmed valve might

indeed have a higher opening pressure, clinically, hypotony were still reported in 5-20

33

% of cases after Ahmed GDD implantation (Coleman AJO 97; Coleman Arch Oph

97).

All the commonly used devices feature a round silicone anterior chamber tube with a

diameter o f between 0.56 mm and 0.63 mm (Table 4). The recommended technique

for insertion is through a paracentesis track created by either a 22 gauge or 23 gauge

(0.65 mm) hypodermic needle. Debate over the optimum needle gauge continues

(Coleman 1995), but insertion often requires considerable manipulation. The resulting

tube to paracentesis fit is often poor and uncontrolled leakage external to the tube

remains common.

Whether due primarily to lack o f internal flow regulation or uncontrolled extrinsic

leakage, problems associated with excessive early aqueous outflow and hypotony

have not been adequately addressed by current GDD designs and implantation

techniques.

1.9.2 Poor Tissue Compatibility

Biocompatibility refers to the ability o f a synthetic material to interface with living

tissues without provoking a detrimental reaction. W ithin the context o f GDD

implantation, suboptimal biocompatibility is manifest in an array o f complications

including early fibrinous occlusion, comeal endothelial failure, tube migration,

extrusion, and fibrous encapsulation leading to filtration failure. Key elements in the

mechanism o f these complications are protein adhesion and micromotion.

34

Elastomeric silicone (polydimethylsiloxane) remains the most commonly used

material in current GDDs. Silicone, PMMA, and other hydrophobic polymers used in

GDDs have a relatively high binding affinity for plasma and interstitial fluid proteins

including albumin, IgG and fibrinogen. These proteins are adsorbed within hours o f

implantation (Pitt et al 1988). Cellular adhesion, cytokine release and chronic

inflammation are thought to be mediated via elements o f this protein film (Tang &

Eaton 1995).

Micromotion, or microscopic shearing o f the implant relative to the surrounding

tissues exacerbates continuing low-grade inflammation. Plate elements o f

contemporary GDDs often impinge on the extraocular muscles, either directly or

through adhesions with septal elements o f the orbital tissues. Even where this does not

produce a frank motility disturbance (Table 6), it is likely that shear forces

transmitted through the relatively rigid materials used in GDD construction produce

significant micromotion. Rabbit experiments in which the Baerveldt GDD was

fenestrated to improve fibrous tissue anchoring, reducing micromotion, demonstrated

a significant reduction in fibrous encapsulation thickness compared with

unfenestrated controls at explantation six months postoperatively (Jacob-Labarre et al

1996). In addition to helping drive subconjunctival fibrosis after GDD implantation,

micromotion transmitted via the tube element o f contemporary GDDs to the anterior

chamber may lead to continuing comeal endothelial cell loss. Superimposed on

perioperative endothelial damage, this is the likely mechanism o f comeal endothelial

failure in association with GDDs.

35

Progressive fibrous encapsulation limits the filtration life o f all contemporary GDDs.

As with poor flow control, suboptimal biocompatibility and the resultant continuing

low grade inflammatory drive to progressive subconjunctival fibrosis have not yet

been adequately addressed.

1.10 Strategies to Improve Success Rates

1.10.1 Plate Surface Area

One strategy for delaying filtration failure has been to increase plate size. In 1981,

Molteno published a series (Molteno 1981) o f 20 patients who received one plate (135

mm^), two plate (270 mm^) or four plate (540 mm^) Molteno implants. Mean post­

operative 1 0 ? ’s were significantly lower for two and four plates compared with single

plate implantation, but did not differ significantly between two and four plates. A

subsequent randomised controlled trial involving 132 patients (Heuer 1992) showed a

higher success rate in the double-plate (270 mm^) Molteno group compared with the

single-plate (135 mm^) Molteno group. A similar trial (Lloyd et al 1994, Britt et al

1999) comparing Baerveldt implants with two different plate areas (350 & 500 mm^)

was less clear cut. Although fewer medications were required for the patients with

500mm^ implants to achieve the target 10? for success (<21 mmHg), some

complications were more frequent with this larger plate size. Overall, a larger

filtration area would appear to improve filtration function (Minckler 1987) at least in

the medium term, but eventual subconjunctival fibrosis over a wider area may

adversely influence the prognosis for repeat GFS.

36

1.10.2 Antimetabolites

In addition to pioneering departures in GDD design, Molteno was amongst the first to

attempt to control subconjunctival wound healing after GFS pharmacologically. He

used steroids, fluphenamic acid and colchicine systemically (Molteno 1983).

Although filtration function appeared to improve, systemic complications and

uncertain benefits lead to the abandonment o f this regime (Minckler 1988).

Successful modulation o f fibroblast function with locally applied antimetabolite drugs

followed however (Skuta 1992; 5-FU Filtering Surgery Study Group 1989), and the

use o f 5-fluorouracil (5-FU Study Group 1989) and M itomycin-C (MMC) (Katz

1995) in conventional GFS has become widespread over the last decade.

In a rabbit study using Baerveldt implants with and without MMC, Prata et al (1995)

were able to show a consistently lower 10? in the MMC treated eyes. The difference

remained statistically significant for up to ten weeks post-operatively. Early clinical

data has been less easy to interpret. A series o f 21 patients who underwent double

plate Molteno tube implantation with adjunctive intraoperative MMC had a higher

success rate (10P<21mmHg) at 12 months than 18 historic controls (68% v 17%)

(Perkins 1995); but the success rate o f only 17% in the control group was unusually

low (Table 3). In another series o f 21 patients (no controls) using intraoperative MMC

and a modified Molteno implant, the success rate (10P<21mmHg) was 76.2% after a

mean follow-up o f 9.4 ± 6.4 months (Susanna 1994).

More recently, a series o f 49 patients with Molteno tube and intraoperative MMC

achieved a one-year success rate o f 76.1% compared with a historical control o f

37

78.7% in 51 patients (Lee 1997). This finding appeared to be confirmed by a

randomised trial, which showed similar lOP, visual acuity and complication rate at 12

months after surgery using pressure-ridged Molteno implants (Cantor 1998).

However, the small sample group in this study (12 MMC and 13 controlled) make it

difficult to draw any definitive conclusion regarding the effect o f MMC on the

success and complications o f GDD implantation.

1.11 Current Indications

High complication rates and the likelihood o f filtration failure within five years have

confined GDD surgery to situations in which trabeculectomy is unlikely to succeed

(Melamed 1990). Improved filtration performance in trabeculectomy with adjunctive

antimetabolite treatment for cases at a high risk o f failure has relegated GDD surgery

still further, and the position relative to other treatment modalities,

(trabeculectomy/MMC or contemporary cycloablation techniques) has not been

clearly defined by clinical trials.

1.12 New Design Concepts

W hilst the post-antimetabolite era o f GFS may have presaged a downturn in the use o f

contemporary GDDs, it also heralds an opportunity to deconstruct some dated design

concepts and move forward.

38

1.12.1 Tube without Plate

The rational basis for using a plate element to physically maintain a drainage reservoir

might be questioned if subconjunctival fibrosis could be controlled pharmacologically

(Kee & Youn 1995). A large surface area o f relatively rigid foreign material may

simply exacerbate the inflammatory stimulus to progressive fibrosis. Realising this, a

number o f investigators are currently re-exploring anterior GDDs (transcleral

implants with no plate element) (Helies 1997; Glovinsky 1997) Clinical results are not

available yet, but success will depend on a marriage o f design and biomaterials

improvements with well controlled pharmacological modulation o f wound healing.

1.12.2 New Biomaterials

Relatively recent advances in biomaterials technology (Ikada 1994; Hall 1989) offer

the prospect o f implanting biologically inert GDDs with rigidity and biointegration

characteristics designed to eliminate micromotion. These new biomaterials should

enhance filtration longevity and prevent modification o f flow resistance by the

accumulation o f biological debris. If these bioinert materials can be incorporated

alongside good dimensional control in manufacture and design departures to prevent

extrinsic leakage, there is no reason in principle why GDDs might not be used in

routine GFS.

39

1.13 Conclusion

The current standing o f GDDs is in some ways analogous to that o f intraocular lenses

in the early seventies, with frequent complications attributable to design and material

inadequacies. Just as improved intraocular lenses have revolutionised cataract surgery

in the recent past, new materials and design departures may transform filtration

surgery with GDDs in the near future.

With the fundamental aim o f developing a new generation o f GDDs suitable for use

as an efficient one-stop intervention in the primary treatment o f glaucoma, the

premises explored in this thesis are: first, that reproducible control over internal flow

resistance and external leakage are possible in a GDD; and second, that new bioinert

materials have the potential to enhance device performance significantly.

40

CHAPTER TWO - Design Changes to Control Flow

Abstract

AIMS.

Firstly, to examine whether small holes produced by 248 nm excimer laser ablation in

a polymer substrate could consistently produce a pressure drop in the desired target

range (5-15 mmHg) at physiological aqueous flow rates for use as a flow restrictor in

a GDD. Secondly, to investigate whether redesigning the GDD exterior to facilitate

introduction and bear evenly on a standard sized slit incision in the eye wall would

reduce external leakage compared to conventional insertion techniques for existing

devices.

M ETHODS.

Single holes with target diameters o f 10pm, 15pm, 20pm and 25pm were drilled

using a 248 nm excimer laser in sample discs (n=6 at each diameter) punched from

75pm thick polyimide sheet. Sample discs were tested in a flow rig designed to

measure the pressure drop across the discs. Using filtered and degassed water at a

flow rate o f 1.4 pl/min. Repeated flow measurements were taken (n=6) for each disc.

After flow testing, all discs were imaged using a scanning electron microscope and

the dimensions o f each hole were derived using image analysis software. In the

external leakage study, corneoscleral buttons (n=13) were prepared from cadaver pig

eyes and mounted on an artificial anterior chamber infused with Tyrode solution.

After the pressure had stabilised, standard occluded silicone tube implants were

inserted through 23G needle stab incisions at the limbus, these were compared against

prototype PM MA implants with a novel shape profile inserted through 1.15 mm width

41

MVR stab incisions at the limbus. The infusion rate was maintained and a second

pressure measurement was taken when the pressure had stabilised. The difference

between the first and second pressure measurement was then compared, as an index o f

external leakage.

RESULTS

Ablated tubes were found to have a near perfect circular outline on both the entry and

exit side. The observed pressure drops across the ablated sample discs at each target

diameter were as follows: 10pm - 25.66±4.9 (mmHg; mean±SD); 15pm - 6.76±1.15;

20 pm - 1.66+1.07; and 25 pm - <0.1 mmHg. The correlation coefficient between

observed pressure drops and those predicted by Poiseuille’s formula was 0.996.

Target ablations o f 15 pm diameter produced tubes that consistently achieved a

pressure drop within the desired range (5-15mmHg). In the external leakage study,

pre-insertion pressures (mmHg; mean ± SD) were 19.00+4.3 (conventional method)

and 20.00+3.9 (new technique with PMMA prototypes). Post-insertion pressures were

significantly reduced (10.40+7.7; p<0.01) for the conventional technique and were

essentially unchanged for the new techniques (18.80+4.9; p>0.1).

CONCLUSIONS

These studies have shown that it is possible in principle to control the dimensions o f a

manufactured tubular lumen in a GDD accurately enough to provide consistent

protection from hypotony in the early period after glaucoma filtration surgery. By

redesigning the external profile o f the device and the incision technique, we have also

shown that it is possible to eliminate uncontrolled external leakage.

42

2.1 Introduction

This chapter will describe a series o f flow experiments to look at ways to improve

flow control in a new generation o f GDDs.

Glaucoma filtration surgery (GFS) involves the creation o f a channel, or fistula,

between the anterior chamber and the subconjunctival space, bypassing the

compromised physiological aqueous drainage pathway through the trabecular

mesh work. Many o f the early postoperative complications o f GFS relate to poor

initial control over aqueous outflow, and safety could be considerably enhanced if

resistance to flow through the drainage fistula (fistular resistanee) were regulated

more effectively.

Early postoperative hypotony (I0P<5mmFIg) is a consistent feature o f unguarded

filtration procedures including laser sclerostomy, and remains eommon after

trabeculectomy both in the immediate postoperative period and after suture release

(Block 1992; 5-FU Filtering Surgery Study Group 1989). Consistent control over

fistular resistance in eonventional GFS is difficult to achieve. The scope for human

error in regulating faetors such as suture tension and flap dimensions in

trabeculectomy, or dissection depth in deep sclerectomy, is considerable. Here we

explore the possibility that a GDD might be used to impart reproducible flow

resistanee in routine GFS.

Existing valved GDDs have been unsuceessful in preventing hypotony in a significant

proportion o f cases (Krupin Eye Valve Study Group 1994; Coleman 1995). Recently

43

examined pressure/flow relationships in leading eurrent devices found that valved

devices tended not to close after initial perfusion (Prata 1995). Simple non-valved

flow resistors (single or multiple small diameter tubes) may therefore be preferable.

Flow resistance in a tubular channel varies with the fourth power o f tube diameter in

accordance with Poiseuille's formula (McEwen 1958). To impart a consistent level o f

flow resistance, tube dimensions (the diameter in particular) must be controlled very

accurately. The required level o f control has never been approached by current laser

sclerostomy techniques (Allan 1993). Far greater accuracy should be possible for laser

ablation o f a homogeneous synthetic polymer substrate in controlled conditions. We

set out to examine whether small tubular holes produced by 248 nm excimer laser

ablation in a polymer substrate could consistently impart a flow resistance in the

desired target range: producing a pressure drop across specimen tubes mounted in an

experimental flow rig o f 5-15 mmHg at physiological flow rates.

Insertion o f conventional GDDs into the paracentesis track produced by a standard

hypodermic needle often requires considerable manipulation. Debate over the

optimum needle gauge (G) continues (Coleman 1995), and hypotony due to external

leakage remains a significant clinical problem. Redesigning the exterior o f the device

to facilitate introduction and improve fit should help prevent external leakage. In a

further series o f flow experiments, external leakage for the tube element common to

existing devices (Table 4; Chapter 1: Page 20) introduced conventionally was

compared with a redesigned GDD and insertion technique.

44

2.2 Fluid Mechanics

If small cylindrical tubes are to be used as the flow resistanee element for a GDD, the

internal dimensions required to provide a given pressure gradient can be calculated

using Poiseuille’s equation if the rate o f production o f human aqueous is known, and

flow through the tube is laminar.

Aqueous flow in normal subjeets during waking hours is thought to be 2.75 ± 0.63

pl/min (Brubaker 1991), but ean be as low as 1.4 ± 0.19 pl/m in (2.3x10’" m^sec'*)

during sleep (Reiss 1984). Sinee the main aim in device design was to provide

eonsistent proteetion from hypotony, the rate o f aqueous flow used in this experiment

was set at 1.4 pl/min, the lowest likely physiological level. The viscosity o f human

aqueous is only slightly greater than water (1.029 compared to 1.000 for water)

(Cavazzani 1909) and for these ealculations, the value o f water was used.

Turbulent flow oeeurs in tubes when the Reynolds number reaches a certain level. At

this point, the pressure gradient aeross the tube will increase markedly as more

mechanical energy is lost. It was therefore essential in GDD design to ensure that

flow remains laminar. It is generally agreed that the lower critical Reynolds number is

2000 above whieh the transition from laminar to turbulent flow oeeurs. Reynolds

numbers can be calculated by using the following formula (Batchelor 2000):

Re = pdu/p

45

WhereRe= Reynolds numberp= density o f aqueous = 1000 kg m'^d ^ tube diameter in metre (10-30 pm)u= average flo w velocity = rate/area = msec^p= viscosity o f the fluid=0.001 Nsecm'^therefore,

Re = kgNsec'^m’’

In Figure 6, Reynolds numbers are plotted against a range o f possible diameters for

short cylinders designed to provide TOP control in the desired range (5-15mmHg) at

physiological flow rates. It is clear that the Reynolds numbers for all the tube sizes

used are well below 2000. Flow in these systems should be laminar (Batchelor 2000).

Figure 6. The relationship between Reynolds number and tube diameter at physiological aqueous flow rate.

0.35

^ 0.25 -

3 0.20

0.10

100 120

Tube Diameter in ^m

46

The application o f Poiseuille’s formula in this context is based on the assumption that

tube length is considerably greater than the diameter. For shorter cylindrical tubes, the

distance required to establish a laminar flow pattern (the entry length) can be

calculated by the following formula:

Entry length = 0.057 x Reynolds Number x hole diameter

Using this formula it can be seen that a laminar flow pattern develops within a short

distance o f entry for all likely flow systems in GDDs. For example, with a 10 pm

diameter hole, the entry length is only 1.6 pm.

Other factors such as abrupt contraction or enlargement o f the tube, and diffuser

effects associated with the entry edges are not significant because o f the low

Reynolds’ number produced by flow systems at physiological aqueous production

rates (Batchelor 2000).

Finally, an estimate o f the diameter required for an ideal tube can be obtained from a

simplified Poiseuille’s formula, as applied by M cEwen (1958) for aqueous outflow.

The pressure drop across the tube is given by:

4Pressure drop = 128. p.I.Q /1 3 6 . Ti.d

Where

ju = aqueous viscosity = 0.001 Nsec/m2 (Cavazzani 1909)/ = length (thickness o f polymer disc used in this study) = 0.075 mm Q = aqueous flo w rate = 1.4 pl/min d = diameter in metre

47

Figure 7a. The pressure drop across a tube versus tube diameter from Poiseuille's formula at a flow rate o f 0.7pl/m in (plain line), 1.4pl/min (dotted line) and 2.8pl/min (dashed line):

Pressure drop = 128//1Q / 1367id^

Where

JU = aqueous viscosity = 0.001 Nsec/m2 I = length = 0.075 mm in this study Q = aqueous flo w rate d = diameter in metres

XBSa.2021

1800 n

1600

1400

1200 -

1000

800 -

600

400

200 -

10 20 30

Tube Diameter (pm)

40 50

48

Figure 7b. I he relationship between tube diameter and length, calculated from the Poiseuille's formula, for a pressure drop across the tube o f 5mmHg at a flow rate o f 0.7pl/min {plain line), 1.4pl/min {dotted line) and 2.8pl/min {dashed line).

.a3f-

70 1

60 -

50 -

40 -

30 -

20 -

T

2 3 4

Length o f Tube (mm)

49

The relationship between pressure drop and diameter o f a tube 50 pm in length is

shown in Figure 7. For example, to obtain a pressure drop o f 5 mmHg, the diameter

o f the tube would be 16.2 pm.

W ith a multiple tube system, the flow rate through each hole will be correspondingly

reduced depending on the total number o f holes. The formula for the pressure gradient

is thus given by the following formula:

4Pressure drop = 128. p.l.Q / 136.N.7i.d

Where

N = Number o f tubes

2.3 Materials & Methods (Internal Flow Control)

2.3.1 Preparation of Polyimide discs & Excimer Laser Ablations

Single holes with target diameters o f 10pm, 15pm, 20pm and 25pm were drilled

using a 248 nm excimer laser (Figure 8) (Exitech Ltd, Oxford UK) in sample discs

(n=6 at each diameter) punched from 75pm thick polyimide sheet (Goodfellow Ltd

Cambridge, UK). Sample discs were mounted in a flow rig (Figure 9) designed to

measure the pressure drop across the discs, using filtered and degassed water as

previously described (Prata 1995). A flow rate o f 1.4 pl/m in was used throughout.

Repeated flow measurements (Figure 10) were taken (n=6) for each disc. Specimen

discs were removed from their holder and remounted between each measurement.

50

Figure 8. A schematic drawing o f the 248 nm (Krypton/Fluorine) excimer laser delivery system used (Model EX-PS-2000, Exitech Ltd, Oxford UK) (not to scale). Beam area at the target surface, and hence ablation diameter, was controlled by the mask aperture. The pulse repetition rate was set at 40Hz and a total o f 100 pulses were delivered to each disc. Specimens with target diameters o f 10 and 15 pm were ablated at a fluence o f 200 mJ/cm^ with mask diameters o f 300 and 450 pm respectively. Those with target diameters o f 20 and 25 pm were ablated at 170 mJ/cm^ with mask diameters o f 600 and 750 pm.

W orkpiece

248 nm Excimer Laser

ProjectionLensC ondenser Lens

M ask H older

51

Figure 9. I 'he tlow rig used was similar to that previously deseribed for flow evaluation in GDDs (Prata 1995). The system eonsisted o f a pressure transdueer (Model P23ID. Gould Inc., California. USA), bridge amplifier (Model M L llO . AD Instruments. New South Wales. Australia) and recorder (Model ML200, AD Instruments. New South Wales. Australia). Pressure changes were recorded using a Mae Lab software (v3.5 AD Instruments. New South Wales, Australia) on a computer (Pertbrma 630. Apple Ltd.. California, USA). A 1 ml hypodermic syringe (Fortuna. Germany) was used with an adjustable electronic syringe pump (Model PHD 2000, Harvard Apparatus. Massachusetts. USA) to control the flow rate. The polyimide discs were held in a 13 mm stainless steel filter holder (Whatman International, Maidstone. UK). A three-way lock was positioned between the water reservoir and the rest o f the system. Silicone tubing (Silicone High Strength Tubing. Altee. Alton. UK) was used throughout.

Water Reservoir

Three-way Lock

ringe on Infusion pump

PressureTransducer

Filter Holder

Computer

52

Figure 10. A typical flow tracing, demonstrating stabilisation o f the pressure recording within approximately 3 minutes o f commencing infusion.

Pressuredrop

(mmHg)

sD

Tim e in m inutes

2.3.2 Flow Rig

The flow rig used was similar to that previously described for flow evaluation in

GDDs (Porter 1997; Prata 1995). The system consisted o f a pressure transducer

(Model P23ID, Gould Inc., California, USA), bridge amplifier (Model M LllO , AD

Instruments, New South Wales, Australia) and recorder (Model ML200, AD

Instruments, New South Wales, Australia). Pressure changes were recorded using a

MacLab software (v3.5 AD Instruments, New South Wales, Australia) on a computer

(Performa 630, Apple Ltd., California, USA). A 1 ml hypodermic syringe (Fortuna,

Germany) was used with an adjustable electronic syringe pump (Model PHD 2000,

Harvard Apparatus, Massachusetts, USA) to control the flow rate. The polyimide

discs were held in a 13 mm stainless steel filter holder (W hatman International,

53

Maidstone, UK). A three-way lock was positioned between the water reservoir and

the rest o f the system. Silicone tubing (Silicone High Strength Tubing, Altec, Alton,

UK) was used throughout. The transducer and the recording system were calibrated

using a water manometer daily prior to experimentation. Once the system was filled

with water, all gas bubbles were flushed out with the disc submerged in water within

the filter holder. At this point, the three-way lock was turned to open the system to

atmospheric pressure and the pressure reading zeroed on the computer. The three-way

lock was then turned to obtain a closed system and infusion pump started. The

pressure reading was allowed to stabilise over 20 minutes and the average reading

from the final 10 minutes was then taken as the pressure drop for the experiment. This

recording was repeated six times for each disc.

Pressure Drop = P 1-P2

Pi = back pressure in mmHg

P2 = atmospheric pressure in mmHg

2.3.3 Scanning Electron Microscopy & Image Analysis

After flow testing, all discs were coated in palladium and imaged using a scanning

electron microscope (JEOL, Akishima Japan). The dimensions o f each hole were

derived using image analysis software (Scion Image, Maryland USA) (Figure 11).

54

Figure 11. 1 he inner edge of the ablated holes viewed at 90° to the specimen surface by scanning electron microscopy were outlined on each side using image analysis software. The diameter a perfect circle of equivalent area was then calculated to estimate entry and exit diameters. Scale bar = 10 pm.

2.4 Materials & Methods (External Flow Control)

2.4.1 Artifleial Anterior Chamber

Using the flow rig described in section 2.3.2, in place o f the filter holder, the silicone

tubing was connected to an artificial anterior chamber (Figure 12) designed to mount

cadaveric porcine corneoscleral buttons. The tension o f the ring holder sealing the

artificial anterior chamber was adjusted until no leakage was seen macroscopieally.

55

Figure 12. Artificial anterior chamber designed to mount cadaveric porcine corneoscleral buttons and the tension of the ring holder sealing the artificial anterior chamber until no leakage was observed. Scale bar = 1 cm.

r

56

2.4.2 Occluded device implantation

Corneoscleral buttons (n=13) were prepared from cadaver pig eyes and mounted on

the artificial anterior chamber (Figure 12) infused with Tyrode solution. The infusion

rate was adjusted to produce a target pressure in the range o f 15-28 mmHg. After the

pressure had stabilised, standard occluded silicone tube implants (0.63 mm external

diameter) were inserted through 23G needle stab incisions at the limbus (conventional

technique), these were compared against prototype PM M A implants with a novel

hape profile (Figure 13) (0.6mm x 1.15 mm) inserted through 1.15 mm width (20G)

A Y R blade (Visitech, USA) stab incisions at the limbus (new technique). The

nfusion rate was maintained and a second pressure measurement was taken when the

iressure had stabilised. The difference between the first and second pressure

neasurement was then compared, as an index o f external leakage. Incisions were

utured and repeated at a different site in such that each implant was inserted in each

:ye with the order o f insertion randomised.

57

Figure 13. This is one embodiment o f a novel glaueoma drainage deviee (patent pending) ineorporating a small diameter hole (A; white cirele) to provide the required fistular resistance and a larger diameter hole (B; dotted circle) temporarily occluded by a thin ablatable membrane. 1 o give the lowest final lOP, resistance is bypassed by ablating this thin membrane using a Y AG laser delivered through a gonioseope once a mature bleb is established. The shape o f this implant is designed to eliminate external leakage after placement through a standardised slit incision. C = external part o f the deviee (Designed by Bruce Allan).

58

2.5 Results

Ablated tubes were found to have a near perfect circular outline on both the entry and

exit side. Observed entry and exit diameters are given in Table 7. All holes exhibited

a slight taper with entry diameters consistently larger than exit diameters.

The observed pressure drop for the specimens examined correlated very closely with

that predicted by a correction o f the Poiseuille’s formula for tapered tubes using the

observed entry and exit diameters (Figure 14). Target ablations o f 15pm diameter

produced tubes that consistently achieved a pressure drop within the desired range (5-

15mmHg). Some variation between repeated flow measurements for the same

specimen was observed (Figure 15).

Table 7. Summary o f results. Target and observed holes’ diameters with the observed pressure drop in each group o f discs. The ’25 pm ’ group all have < 1 mmHg pressure drop.SD = standard deviation

Target Hole Diameter Observed Hole Diameter Observed Pressure Drop

(pm) (Mean ±SD pm) (Mean ± SD mmHg)

10 12.61 ± 0.05 (Entry side)

11 ± 0.05 (Exit side)25.66 ±4.9

15 16.67 ± 0.82 (Entry side)

15.1 ±0.25 (Exit side)6.76±1.15

20 24.32 ± 1.48 (Entry side)

21.26 ± 0 .3 (Exit side)1.66 ±1.07

59

Figure 14. Observed pressure drop versus predicted. Due to the tapering nature o f the laser holes, the fbllo\ving modification o f the formula was used:

Pressure drop glQ17%

Ri“+R|R2+R23R/R^

Wheren = aqueous viscosity ^ 0.00 i Msec/m2 I = length = 0.075 mm in this study Q = aqueous flo w rate ^ 1 . 4 yl/m in in this study Rj = Radius on one side in metres R2 = Radius on another side in metresNote the orientation in which the disc was tested would not make any difference to the final pressure drop.

35 1

-#— Predicted Pressure Drop Observed Pressure Drop3 -

I 2 5 -

'10 pm’ '15 pm' '20 pm' '25 pm'

Intended Hole Diameter (pm)

60

Figure 15. The pressure drop across 5pm tubes o f 75pm length was consistently within the desired target range (5-15m mHg) at a flow rate o f 1.4pl/min. Some variation between specim ens attributable to dimensional variation was observed along with some variation between m easurem ents (n=6; SD shown above by error bars) for the same specimen.

12 1

0 1 2 3 4 5 6

Disc Number

61

In the external leakage study, pre-insertion pressures (mmHg; mean ± SD) were

19.00±4.3 (conventional method) and 20.00±3.9 (new technique with PMMA

prototypes). Post-insertion pressures were significantly reduced (10.40±7.7; p<0.01)

for the conventional technique and were essentially unchanged for the new technique

(18.80±4.9; p>0.1) (Figure 16) PM M A prototypes were considerably easier to insert

than conventional silicone tubing.

62

Figure 16. Graph showing the mean pre-insertion and post-insertion pressures (error bars = SD) for standard occluded tubes and prototypes with the modified external cross-section. Post-insertion pressures were significantly reduced (10.40±7.7; p<0.01) for the conventional technique and were essentially unchanged for the new technique.

Pre Post Pre Post

25

nin

Hg

20

15

10

IVIVR P a ra cen tes is & P ro to ty p e 23G P a ra c en tes is & C o n v en tio n a l T u b e

63

2.6 Discussion

Leading current GDDs have been developed with little available data to substantiate

manufacturers’ claims for flow performance (Krawczyk 1995) or biocompatibility.

Recent evaluation has exposed design flaws (Lee 1998), poor quality control in

manufacture (Porter 1997), and poor flow control (Prata 1995). To gain acceptance as

a method o f preventing hypotony in routine GFS, a new GDD must demonstrate

consistent control over internal flow (flow through the GDD), good protection from

external leakage (leakage between the exterior o f the GDD and the sclerocomeal

tissues through which it is implanted), and excellent biocompatibility. We have

adapted existing methods o f evaluating GDD flow performance in an examination o f

internal flow control using single small diameter tubes as flow resistors.

The good fit between the observed pressure drop across specimen tubes and that

predicted by the Poiseuille's formula was an expected finding. Poiseuille's formula is

applicable where laminar flow exists in a fluid o f uniform viscosity contained within a

rigid tube. Uniform viscosity can be assumed for aqueous, and the likelihood o f

turbulence within a system increases with flow velocity. Physiological flow rates are

sufficiently slow that laminar flow would be established within 2pm o f tube entrance,

and edge effects would be insignificant. Low level lumenal elasticity would be

tolerated in any GDD system using small diameter tubes as flow restrictors.

Polyimide is widely available, inexpensive, and absorbs light strongly at 248nm.

Relatively thin sheets (75 pm) were used to facilitate accurate ablation, and to allow

for the possibility o f subsequent perforation using a Nd:YAG laser to abolish fistular

64

resistance. The logic here is that fistular resistance would only normally be required in

the early postoperative period, prior to the development o f a mature conjunctival

filtration bleb and adequate resistance to flow distal to the GDD. A <20pm hole could

block even with a single macrophage and is unlikely to be practical as a flow resistor

in vivo. Longer tubes imparting equivalent fistular resistance would allow the use o f

larger diameters (Figure 7b), and could be incorporated alongside a larger diameter,

low resistance tube guarded by a thin Y AG ablatable membrane in a GDD with

reversible fistular resistance (Figure 13).

The observed variation between flow measurements for given specimens (Figure 15)

may have been attributable to minor accretions o f debris within the tube lumens

during specimen handling. This highlights the importance o f using a material that

would resist protein conditioning and cellular adhesion in vivo. In the following

chapter we explore the use o f phosphorylcholine (Hall 1989) based polymers in this

context.

The excimer laser system used was non-dedicated. Improvements in laser delivery are

possible that would enhance accuracy and reduce the taper produced by a convergent

beam. Extrusion and moulding technologies may also be applicable, particularly

where longer tubes are used. Even with the system used, ablation was considerably

more accurate than for laser sclerostomy (Allan 1993).

The needle used to form the paracentesis tract in conventional GDD implantation is

either a 22G or 23G (Coleman 1995). Our study o f protection from external leakage

has shown that, even through the smaller 23G paracentesis tracts, insertions were

65

liable to cause significant leakage in some eyes. In addition to protecting from

external leakage effectively, our shape modifications and the use o f a standardised

MVR blade incision reduced the effort and manipulation required for device insertion

considerably.

2.6 Conclusions

We have shown that it is possible in principle to control the dimensions o f a

manufactured tubular lumen in a GDD accurately enough to provide consistent

protection from hypotony in the early period after GFS. We have also demonstrated

good protection from external leakage using a prototype device with a modified

external profile designed to bear evenly on a standardised slit incision.

66

CHAPTER THREE - Biocompatibility Studies

Abstract

Aim

We set out to compare phosphorylcholine coated polymethyl methacrylate (PC-

PMMA) with materials used in existing and experimental glaucoma drainage devices

in a series o f in vitro assays reflecting key dimensions o f biocompatibility in the

context o f glaucoma drainage device implantation.

Methods

Sample discs o f PC-PMMA and control materials (silicone, PMMA, polypropylene,

and PTFE) were exposed to fibrinogen, fibrin, human tenons capsule fibroblasts,

human macrophages and a conjunctival bacterial isolate (staphylococcus epidermidis)

in standardised quantitative assays designed to measure the adherent biomass. Areas

o f damage to cultured monolayers o f bovine comeal endothelial cells produced by

standardised direct contact with test material discs were also measured.

Results

Fibrinogen and fibrin adhesion to PC-PMMA were significantly lower than uncoated

PM M A (p=0.004). Phosphorylcholine coating o f PMMA also significantly reduced

the adhesion o f human scleral fibroblast (p=0.002), bacteria (p= 0.002) and

macrophage (p=0.01) compared with uncoated PMMA. All the other biomaterials

showed either similar or insignificantly different levels o f adhesion to all the proteins

and cells tested, except for polypropylene, which had a significantly higher level of

bacteria adhesion than PMMA (P = 0.026). PC-PMMA also had the lowest area o f

endothelial damage.

67

Conclusion

Phosphorylcholine coating is a new material technology that offers considerable

promise in the field o f glaucoma drainage device development.

68

3.1 Introduction

The high complication rates associated with existing GDDs have restricted their use to

circumstances in which trabeculectomy is unlikely to succeed (Melamed 1990).

Although laser technologies and new design concepts described in Chapter Two,

suggest the possibility o f using GDDs to provide consistent flow control in routine

glaucoma filtration surgery, acceptance would require an improvement in the

biocompatibility o f materials used in construction. In particular, since the internal

dimensions o f a GDD flow pathway could quickly be modified in vivo by biological

spoliation, leading to marked and uncontrolled increases in flow resistance, a material

which resists non-specific protein and cellular spoliation effectively would be vital.

This chapter will highlight the problems associated with poor biocompatibility in the

context o f GDDs and examine the biocompatibility indices o f a new bioinert material.

Biocompatibility refers to the ability o f a synthetic material to interface with living

tissues without provoking a detrimental reaction. W ithin the context o f GDD

implantation, suboptimal biocompatibility is manifest in an array o f complications

including tube occlusion, endophthalmitis, comeal endothelial failure, tube migration,

extrusion, and fibrous encapsulation leading to filtration failure. Key elements in the

mechanism o f these complications are protein adhesion and micromotion.

Elastomeric silicone (polydimethylsiloxane) remains the most commonly used

material in current GDDs. Silicone, polymethyl methacrylate (PMMA),

69

polypropylene (Figure 17) and other hydrophobic polymers used in GDDs have a

relatively high binding affinity for plasma and interstitial fluid proteins including

albumin, IgG and fibrinogen. These proteins are adsorbed within seconds o f

implantation (Pitt et al 1988). Cellular adhesion, cytokine release and chronic

inflammation all occur as secondary events following the deposition o f a conditioning

protein film (Tang & Eaton 1995).

Bioinert materials are designed to resist protein and cell adhesion non-specifically,

minimising inflammatory responses, and preventing occlusion in implants used in

fluid contacting environments (Allan 1999).

70

Figure 17. Polymers commonly used in ophthalmology.

C = 0 Polymethyl meethacrylate (PMMA)

Silicone

[-CH2-C-]-

CH2OH

[-CH2-CH-]

Poly(2-hydroxyethyl methacrylate) [polyHEMA hydrogel]

Polypropylene (PP)

-[-CF2-CF2-]- Poly(tetrafluoroethylene)

71

3.2 Bioinert Materials

3.2.1 Heparin Coating

Covalently linked heparin surface modification (Figure 18) has been used as a

strategy to reduce cellular spoliation and inflammation associated with intraocular

lens implantation after cataract surgery (Larsson 1989). In comparison with uncoated

PMMA, reported clinical benefits include reduced post-operative inflammation

(Starvou 1994) and less giant cell spoliation (Trocme 2000). In vitro results suggest

less bacterial (Portoles 1993) and lens epithelial cell (Majima 1996) adhesion.

Heparin prevents thrombus formation by binding to antithrombin III (ATIII) and

catalysing the action o f ATIII to neutralise thrombin, thus preventing catalysis and

cleavage o f fibrinogen to fibrin.

Although heparin coating allows PMMA to become more bioinert, modem alternative

lOL materials, including dimethyl siloxane (silicone) and 2-hydroxyethl methacrylate

HEMA have proved to be superior in both in vivo (Ravalico 1997) and in vitro (Joo

1992, M ajima 1996). Most contemporary GDDs use elastomeric silicone for at least

the tube component.

72

Figure 18. A schematic representation o f heparin immobilized onto the surface o f a polymer substrate using hydrophilic spacer groups.

HepariiHeparii

Heparii

Hydrophilic spacer groups

Substrate

73

3.2.2 Surface Passivation

In contrast to the use o f a molecule such as heparin with a specific biological action to

prevent biological spoliation, surface passivation refers to the process o f making a

surface unreactive by coating with a thin layer o f a relatively unreactive material.

Polymer surfaces passivated by albumin have been shown to reduce occlusion in

extracorporeal circulation systems (Mulvihill 1990) and to reduce the level o f platelet,

neutrophil, and macrophage adhesion (Tang 1993) compared with unmodified control

materials.

Early in vitro studies indicated reduced endothelial cell damage on contact with

surface passivated intraocular lenses (Balyeat 1989). However, this optimism was not

translated to later clinical (Umezawa 1993, Amon 1996, Ravalico 1997), and

experimental studies (K ochounianl991, Majima 1996, Lawin-Brussel 1992), and

albumin surface passivation has not been pursued as a strategy for rendering

ophthalmic implant materials bioinert.

3.2.3 Hydrogels

Hydrogels are cross-linked polymeric structures with high water content (Figure 17).

A hydrogel in routine ophthalmic use as a soft contact lens material and in intraocular

lenses is HEMA (Barrett 1987). HEMA is closely related to PMMA. Synthesis

involves the adding o f a methylol group to the common methyl methacrylate side

group. This addition results in an extremely hydrophilic polymer (HEMA). Increased

74

hydration appears to reduce protein and cell spoliation, but protein deposition remains

a significant clinical problem for HEMA contact lenses, and HEMA has not been

widely adopted as an lOL material (Allan 1999). Suitability for use in GDDs has been

thrown into doubt by reports o f hydrogel fragmentation (D ’Hermies 1998) and

extensive cell adhesion (Ayyala 2000) when placed sub-conjunctivally.

3.2.4 Polyethylene Oxide Polymers

Polyethylene oxide polymers (PEG) are another potentially useful group o f bioinert

materials, which loosely reflect some o f the properties o f natural mucous membrane

surfaces. PEG polymers are highly hydrophilic, mobile, long chain molecules, which

trap a large hydration shell. They enhance resistance to protein and cell spoliation

when grafted onto a variety o f material surfaces (Kim 1996, Cao 1996). PEG

polymers are also amenable to end group coupling for surface immobilisation o f

biologically active molecules such as heparin to add specific functionality (Cao 1996).

3.2.5 Biomimetic bioinert materials

Increasingly, biomaterials solutions are sought through mimicking or incorporation

natural structures in synthetic materials (Allan 1999). This biomimetic approach has

been adopted in the synthesis o f a new class o f polymers based on phosphorylcholine

(PC) (Figures 19-21). PC is the hydrophilic head group o f the predominant

phospholipids (lecithin and sphingomyelin) in the outer envelope o f mammalian cell

membranes (Hayward 1984; Durrani 1986; Kojima 1991). PC polymers are designed

to approximate the chemical properties o f the natural cell wall, and in particular the

75

ability to resist non-specific protein attachment. As coatings or bulk materials, these

biomimetic bioinert polymers are thought to have enhanced resistance to non-specific

protein adhesion. Early applications o f this technology have included chest drains

(Hunter 1993) and contact lenses (Knzler 1994).

76

Figure 19. A schematic representation o f the mammalian cell wall. Highly hydrated phosphorylcholine head groups (small spheres) are orientated externally in this self­assembling bilayer. Biocompatibles PC technology is based on the observation that the outside leaflet o f a red blood cell membrane is haemocompatible, while the inner leaflet is thrombogenic.

Figure 20. The inner, thrombogenic surface contains negatively charged phospholipids while the outer bilayer is composed o f zwitterionic phospholipids. These have both positive and negative charges in close proximity but are electrically neutral overall.

77

Figure 21. The phosphorylcholine (PC) coating mimics the zwitterionic structure of

natural membranes, thereby conferring properties of haemocompatibility. (A) The PC

headgroups bind large numbers of water molecules tightly around them, making it

energetically unfavourable for other materials to interact with the PC surface. (B)

Further away the water molecules are bound less strongly, sliding over one another

resulting in a low coefficient o f friction.

78

3.3 Biocompatibility in the Context o f Glaucoma Drainage Devices

Key dimensions o f biocompatibility in relation to GDD implantation are: resistance to

non-specific biological spoliation and tube occlusion, resistance to fibrotic

encapsulation and drainage failure (foreign body reactions), reduced contact

associated corneal endothelial damage, and resistance to bacterial adhesion. Each o f

these elements is considered below.

3.3.1 Protein & Cell Adhesion - Tube Occlusion

Valve mechanisms in glaucoma drainage devices (GDDs) have largely failed to

provide consistent flow control and protection from hypotony. Limitations o f

currently marketed valved devices have recently been exposed in theoretical (Lee

1998) experimental (Prata 1995; Porter 1997) and clinical examinations (Lim 1998).

Flow control in non-valved devices is critically dependent on the internal dimensions

o f the flow passage. For tube devices, resistance varies with the fourth power o f tube

diameter in accordance with Poiseuilie's law (McEwen 1958). Small changes in

diameter induced by protein or cellular spoliation may lead to substantial changes in

flow performance. Resistance to surface spoliation is therefore a key requirement for

materials used in GDDs which seek to control flow using single or multiple small

bore tubes as flow resistors.

79

Protein and cellular components, which are likely to play an important role in tube

surface spoilation in the post-operative aqueous environment, include fibrinogen,

fibrin, and macrophages. Small-bore filtration channels may also be capped off

externally by scleral fibroblasts growing into a fibrin scaffold (Shahinian 1992).

3.3.2 Protein and Cell Adhesion - Chronic Inflammation & Bleb Fibrosis

As with all implanted biomaterials, GDDs stimulate a foreign body reaction and a

variable degree o f chronic inflammation. In the context o f GDD implantation, this

chronic inflammatory process will lead to progressive fibrous encapsulation. This

chain o f events limits the filtration life o f all contemporary GDDs (Table 5; Chapter

1: Page 27).

In vivo studies using rabbits have suggested that both physical and chemical

properties o f biomaterials may determine the level o f inflammation (Ayyala 2000)

and consequent fibrous encapsulation thickness (Jacob-Labarre et al 1996) related to

subconjunctival implants. Although the mechanism o f foreign body reactions is

incompletely understood, it is likely that fibrinogen adhesion is important in the

initiation o f inflammatory responses (Tang 1993). Adherent macrophages may play a

major role (Schoen 1996) in orchestrating the cytokine drive to chronic inflammation.

80

3.3.3 Cell Damage - Endothelial Failure

Comeal endothelial failure is a common complication GDDs implantation (Chapter

1), occurring in up to 34% o f cases after a mean follow up o f two years in GDDs

implantation after previous comeal graft surgery (Beebe 1990).

The mechanism for endothelial failure after GDD implantation is not fully

understood. Continued small movements o f the tube element relative to the com ea

(micro-motion) transmitted from repeated minor displacement o f the GDD plate

element during eye movement may be important in accelerating endothelial loss. This

gradual attrition may be superimposed on significant intraoperative endothelial

damage, and damage in the early postoperative period from macroscopic contact

between the implant and the com ea in association with poor tube positioning or

anterior chamber shallowing.

Each o f these possible elements o f the mechanism for endothelial failure after GDD

implantation involves contact between the endothelium and the implant. Studies o f

comeal endothelial cell damage resulting from standardised direct contact with

intraocular lenses have shown variation between materials (Kassar 1980, Barrett

1984, Mateo 1989, Balyeat 1989). A material for which contact induced endothelial

damage is relatively low should help reduce endothelial loss in association with GDD

implantation.

81

3.3.4 Bacterial Adhesion - Endophthalmitis

Endophthalmitis associated with GDD is a rare but serious complication, with an

overall incidence o f less than 0.5% (Table 6; Chapter 1: Page 29). The possible

routes o f bacterial entry include; contaminated surgical instruments or GDD and GDD

erosion. The initial adhesion o f bacteria to polymer substrata is a complex process but

can be assumed to be dependent upon the host environment, materials and the type o f

cells involved (Gristina 1996). Once bacteria are organised within a ‘biofilm ’

(Gristina 1987), they become relatively resistant to both antibiotics and host defence

mechanisms.

3.4 Aims

We set out to compare PC-coated polymethyl methacrylate (PMMA) with materials

used in existing and experimental GDDs (Table 3; Chapter 1: Page 18) in a series o f

in vitro assays reflecting key dimensions o f biocompatibility in the context o f GDD

implantation.

3.5 Materials & Methods

3.5.1 Preparation of Discs

Sample discs (8mm diameter) were punched from 0.5mm thick sheet PMMA cast

from polymer beads (ICI, London, UK), polypropylene (Goodfellow, Cambridge,

UK), PTFE (Goodfellow, Cambridge UK), and silicone (Goodfellow, Cambridge,

82

UK). Additional PMMA discs were eoated with P C I008 (2:1 copolymer o f lauryl

methacrylate and methacryloyl phosphorylcholine) (Biocompatibles, Famham UK).

Sample discs were washed sequentially with a 7X-PF detergent (ICN

Pharmaceuticals, Hants. UK) and distilled water. The coated discs were then air-dried

in sterile conditions for 10-15 minutes at room temperature prior to experimentation.

Six dises o f eaeh material were used for each o f the adhesion assays.

3.5.2 Cell and Protein Adhesion

3.5.2.1 Fibrinogen Adsorption

I'^^-labelled fibrinogen (ICN Pharmaceutical, Hants. UK) was diluted (ratio 1:9) with

unlabelled human fibrinogen (Sigma, Dorset, UK) in 12.6ml o f phosphate buffered

saline (PBS). A total o f 25.2ng o f fibrinogen in 40pl o f solution was pipetted onto

each disc in a 24 well plate and incubated at room temperature for 2 hours. The discs

were then washed (x3) in PBS, transferred to scintillation vials and submerged in

scintillation fluid. The scintillation count for each vial was then measured over one

minute in a Liquid Scintillation Counter (Model 1209 Rack Beta, LKB Ltd., USA).

3.5.2.2 Fibrin Adhesion

Fresh venous blood (20ml) was obtained from a healthy adult male volunteer in a

citrated vial to prevent clotting. The blood was centrifuged for 10 minutes at 3000

rpm to obtain plasma. 0.1ml o f plasma was diluted with 1ml o f normal saline. 0.1ml

o f 0.025M calcium chloride solution was then added to this mixture to initiate

83

clotting. 0.2ml o f this solution was immediately pipetted onto centre o f each sample

disc. These discs were incubated for 2 hours at room temperature to allow the

formation and adherence o f fibrin, before being washed (x3) in PBS and transferred to

a sterile 24 well plate. Fibrin deposits were dissolved by adding 0.2ml o f 4M

guanidine thiocyanate into each well with constant agitation (30 rpm) for 1 hour at

room temperature. The tyrosine residue in 0.1ml o f this solution was measured as a

quantification marker for dissolved fibrin using a spectrophotofluorometer (Model

7687, American Instrument Company, Maryland, USA) (excitation wavelength

275nm; emission wavelength 310nm).

3.5.2.3 Human Scleral Fibroblast Adhesion

Eye bank scleral rim tissue from adult donors (Moorfields Eye Hospital Eye Bank)

was cultured to produce a strain o f scleral fibroblasts in Eagle’s minimal essential

medium (MEM) (Gibco, Life Technology Ltd., Paisley, UK) supplemented with 10%

foetal calf serum, 1% penicillin and 1% streptomycin. Cells were serially passaged to

6-7 population doublings by standard trypsin-EDTA dispersion. The fibroblasts were

then seeded in MEM at a density o f 6000 cells cm'^ into wells containing the sample

discs, and incubated at 37°C under 5% CO2 to allow attachment for 48 hours. The

discs were then washed (x3) in PBS and transferred to a new 24 well plate.

Intracellular ATP was liberated by incubating each disc with 100 ml o f sterile

hypotonic lysis buffer (0 .0IM Tris-Acetate pH8, 2mM EDTA). After 30 minutes the

ATP solution was diluted 1:1 with an additional buffer (0.2M Tris-HCl, 0.2mM

EDTA, pH 7.3). ATP levels in this buffered lysate were then measured as a

quantification marker for the adherent fibroblasts by ATP dependent luminometry.

84

using a commercial luciferase test kit (BioOrbit-W allac, Turku, Finland) and a 96

well plate luminometer (Amersham, Bucks. UK).

3.S.2.4 Macrophage Adhesion

M ononuclear cells were purified using commercial gravity separation media as

described below. Fresh venous blood was obtained from a healthy 32 year-old male

volunteer. The blood was defibrinated by agitation in a vial containing sterile glass

beads for 5 minutes. The blood was then diluted 1:1 with Macrophage Serum Free

Medium (RPMI1640 medium, Gibco, Life Technology Ltd., Paisley, UK) and slowly

pipetted into a tube containing a purification medium (LymphoSep, ICN-

Pharmaceuticals, Hants. UK). The mixture was centrifuged at 400g for 20 minutes to

separate the cells. The central layer containing the mononuclear cells was sampled.

The mononuclear cells were then pelleted by a further centrifugation at 400g for 10

minutes. The mononuclear cells were re-suspended in RPMI1640 medium and

counted using a haemocytometer. Sample discs were placed in a 24 well tissue culture

plate and 2x10^ cells in 0.5 ml o f media are pipetted onto each disc in sequence. The

plates were incubated for 24 hours in a 37°C incubator under 5% COi.The discs were

then washed (x3) with PBS and fixed in 4% paraformaldehyde for 10 minutes at room

temperature. The fixed cells were then permeabalised by incubation with a 1%

solution o f Triton X I00 (Sigma, Dorset, UK) for 2 minutes, and macrophages

detected using the protocol described below using an Avidin-Biotin-Peroxidase kit

(RapidStain, Sigma, Dorset, UK).

85

Endogenous peroxidase activity in the cells was quenched by 72 hours incubation in

9% H2O2 and repeated washing in PBS. Mouse anti-human macrophage (CD68)

monoclonal antibody (Dako Ltd. Bucks. UK, diluted 1:100 in PBS + 1% Foetal C alf

Serum) was added to each disc and allowed to incubate overnight at 4°C in a

humidified chamber. The discs were then washed (x3) in PBS and macrophages were

observed using a high affinity biotin conjugated anti-mouse antibody and an avidin-

peroxidase detection system with AEG as a chromogen. The average number o f

macrophages (stained red) was determined in 50 randomly selected fields under light

microscopy.

3.5.2.5 Data Analysis

For each o f the cell adhesion assays above, results are presented graphically as box-

plots showing the 10̂ *̂ , 25̂ *̂ ’ 50^ '̂ and 90^ ̂ centiles as vertical boxes with error

bars. Statistical analysis was performed using SPSS for W indows software (SPSS

Inc., Chicago, USA). The Mann-W hitney non-parametric test was used to detect any

statistically significant differences between uncoated PM MA and the other test

materials.

3.5.3 Endothelial Damage

A commercially available strain o f BCE cells (Strain CRT-2048, American Type

Cultural Collection, New Jersey, USA) was purchased and seeded in 24 well culture

plates in D ulbecco’s Modified Eagle’s Medium (Gibco, Life Technology Ltd.,

Paisley, UK) supplemented with 10% foetal calf serum and 5% foetal bovine serum.

The BCE cells were grown to confluence at 37®C in 5% CO2 with a change o f media

86

every two days. Before each contact assay, media were aspirated from the

monolayers, which were rinsed with Im L o f fresh medium to remove all unattached

cells.

13 mm diameter discs o f silicone, PMMA, PTFE, polypropylene and PC-PMMA

(n=6) were gently lowered onto the BCE monolayers using a vacuum suction device

with a 2.4 g o f titanium weight placed on top o f each disc. The weights and discs were

removed using a pair o f forceps and a vacuum suction device after 5 minutes o f

contact. A 0.4% trypan blue solution was used to stain damaged cells.

After staining, the central area o f each plate was photographed at low magnification

under a light microscope. Digital photographs were then analysed and areas with

staining and stripping o f cells were outlined. The total area o f damage in each well

(including the area stripped o f cells) was measured using image analysis software

(Scion Image, M aryland USA) and expressed as a percentage o f the total disc area to

derive a damage coefficient for each material.

Analysis o f variance (ANOVA) using the Bonferroni method was performed using

SPSS for Windows software (SPSS Inc., Chicago, USA) to compare endothelial

damage coefficients between materials.

3.5.4 Bacterial Adhesion

Staphylococcus Epidermidis strain 901, a clinical isolate, was grown in liquid culture

in Oxoid No. 2 broth at 37^C in 5% CO2. Bacterial cells were pelleted in a refrigerated

87

centrifuge and washed three times in sterile PBS. The cells were resuspended in PBS

using optical density measurements to standardise bacterial cell concentration at

3x10* cell m l''.

One cm^ discs o f the test biomaterials were incubated with the resultant bacterial

suspension for four hours with constant agitation. The discs were then washed (x3) in

PBS and transferred to a new 24 well plate.

Intracellular ATP was liberated by incubating each disc with 100 ml o f sterile

hypotonic lysis buffer (0.0IM Tris-Acetate pH8, 2mM EDTA). After 30 minutes the

ATP solution was diluted 1:1 with an additional buffer (0.2M Tris-HCl, 0.2mM

EDTA, pH 7.3). ATP levels in this buffered lysate were then measured as a

quantification marker for the adherent bacteria by ATP dependent luminometry, using

a commercial luciferase test kit (BioOrbit-W allac, Turku, Finland) and a 96 well plate

luminometer (Amersham, Bucks. UK).

Results are presented graphically as box-plots showing the 10‘*̂, 25**̂ ’ 50̂ *̂ ’ 75* ,̂ and

90̂ '̂ centiles as vertical boxes with error bars. Statistical analysis was performed using

SPSS for Windows software (SPSS Inc., Chicago, USA). The M ann-W hitney non-

parametric test was used to detect any statistically significant differences between

uncoated PMMA and the other test materials.

88

3.6 Results

3.6.1 Proteins & Cell Adhesion

Fibrinogen adsorption is illustrated in Figure 22. Only PC-coated PMMA discs

reduced the amount o f fibrinogen adsorption significantly (p=0.004), relative to

PMMA. Figure 23 shows fibrin adhesion on the test materials. Fibrin adhesion to the

PC-coated PMMA discs was minimal, and significantly lower than un-coated PMMA

(p=0.004), with other materials exhibiting similarly elevated fibrin adhesion levels.

PC coating the PMMA discs also modified the cellular adhesion profiles and these

results are shown in Figures 24 and 25. The reduction in human scleral fibroblast

adhesion to PC-coated PMMA was statistically significant (p=0.002) compared to un­

coated PMMA discs (Figures 24). The adhesion o f human scleral fibroblasts to

silicone, polypropylene and PTFE discs was not significantly reduced in comparison

with PMMA (Figure 24). The adhesion o f human macrophages was also significantly

reduced by PC coating o f PMMA (p=0.01) (Figure 25). Although both the PP and

PTFE discs have a higher mean number o f macrophages adherent per field in

comparison with un-coated PMMA discs, these differences are not statistically

significant (p=0.057).

3.6.2 Bovine Endothelial Cell Damage

Percentage areas o f damage (mean ±SD) after standardised contact were as follows:

PMMA 5.95+4.60%; polypropylene 1.63±0.81%; PTFE 1.00±0.19%; silicone

89

3.08+2.68% and PC-PMM A 0.15±0.25% (Figure 26). The uncoated PMMA discs

tested caused considerable damage, evidenced by the marked stripping o f endothelial

cells (Figure 27). Significant endothelial cell disruption was also a feature o f contact

with silicone specimen discs. Only PC-PMMA discs had a level o f damage (p=0.04)

significantly lower than that observed for uncoated PMMA.

3.6.3 Bacterial Adhesion

The mean light unit o f bacteria adhered to polymer surfaces were as follows: PMMA

0.33 ± 0.32, PC-PMM A 0.02 ± 0.01, silicone 0.37 ± 0.03, PTFE 0.30 ± 0.19 and

Polypropylene 4.06 ± 3.2. %. Results are presented graphically as box-plots showing

the 10*'\ 25‘̂ ’ 50̂ *̂ ’ 75‘'\ and 90*'’ centiles as vertical boxes with error bars (Figure 28).

The uncoated PMMA disc tested had considerable more bacteria adhered to its

surface compared with PC-coated PMMA disc (Figure 29). Only PC-PMMA discs

had a significantly lower level o f bacterial adhesion (p= 0.0.002) than other test

materials. Polypropylene discs had significantly higher levels o f bacterial adhesion

compared to PMMA (p=0.026).

90

Figure 22. Adsorption o f Iodine’"^-labelled fibrinogen to biomaterials after 2 hours incubation in PBS. Only PC-coated PM M A discs reduced the amount o f fibrinogen adsorption significantly (p=0.004), relative to PMMA.

CD

9000

8000

7000

CDp- 6000

J 5000bo

pcon

4000

3000-

W -

PMMA PC-PMMA Silicone PTFE PP

Materials

91

Figure 23. Adhesion o f fibrin to biomaterials from fresh plasma after 2 hours incubation. The level o f fibrin adhesion to PC-coated PMMA discs was minimal, and significantly lower than un-coated PMMA (p=0.004).

PMMA PC-PMMA Silicone

M ateria ls

92

Figure 24. In vitro adhesion o f human scleral fibroblast to biomaterials. The reduction in human scleral fibroblast adhesion to PC-coated PM M A was statistically significant (p=0.002) compared to un-coated PMMA discs.

40

30

20

10

0

I_____

PMMA

‘i

PC-PMMA Silicone PTFE PP

M ateria ls

93

Figure 25. In vitro adhesion o f macrophage to biomaterials. The adhesion o f human macrophages was significantly reduced by PC coating o f PMMA (p=0.01 )

I 10

PMMA PC-PMMA Silicone PTFE

M ateria ls

94

Figure 26. Mean percentage damage to the bovine corneal endothelial monolayer culture alter standardised contact with different biomaterials. PC-PM M A discs had a level o f damage (p=0.04) significantly lower than that observed for uncoated PMMA.

01CQE

Q

OX)CQ

CQJUimQJ

a .

P M M A P C - P M M A Si l i cone PP

Materials

P T F E

95

Figure 27. Light microscopy photographs of typical damage to the bovine comeal endothelial monolayer culture after contact with PMMA and PC-coated PMMA. The red dashed line dividing the area A, which had stripped endothelial monolayer, from area B, which showed relatively intact endothelial monolayer. Scale bar = 10 pm.

PMMA PC-PM M A

96

Figure 28. In vitro adhesion o\'Staphylococcus Epiderm idis to biomaterials. Only PC- PMMA discs had a significantly lower level o f bacterial adhesion (p= 0.0.002) than other test materials. Polypropylene discs had significantly higher levels of bacterial adhesion compared to PMMA (p=0.026).

P M M A P T F E Si l i cone PP

MaterialsP C - P M M A

97

Figure 29. Scanning electron microscopy of PMMA and PC-coated PMMA discs showing the typical level of bacteria adhesion. A = adhered colonies of Staph, epidermidis. Scale bar = 10 pm.

PMMA disc

PC-PMMA disc

98

3.7 Discussion

3.7.1 Choice o f Test Biomaterials

PC coatings have been developed specifically to enhance biocompatibility for

materials used in fluid contacting applications. We chose PC-coated PMMA as a

candidate material for our novel GDD. Polypropylene and silicone are the most

common biomaterials used in the construction o f current GDDs (Table 4; Chapter 1;

Page 20). We also included PTFE, as it had been proposed as a potential biomaterial

for the construction o f GDDs (Helies 1997).

3.7.2 Cell & Protein Adhesion

Implanted polymeric materials quickly adsorb proteins from the surrounding fluid or

tissue environment. Adsorption occurs via a range o f bonding mechanisms, and

results in a biologically conditioned surface with modified interfacial properties. Cells

interact predominantly with this modified surface rather than the material surface

itself (Tang 1995). The biological adhesion characteristics o f a material are therefore

governed by its protein adsorption properties.

Fibrinogen adsorption may be o f particularly importance. In addition to its role as a

fibrin precursor, fibrinogen appears to be pivotal in establishing inflammatory

responses to implanted synthetic polymers (Tang 1993). Histological studies in

complement or immunoglobulin depleted mice have revealed inflammatory responses

similar to non-depleted controls (Tang 1993). In contrast, fibrinogen depleted mice

99

did not mount an inflammatory response unless the test material was pre-coated with

fibrinogen (Tang 1993). Fibrinogen is also thought to promote cell adhesion by

integrin binding to an RGD m otif similar to fibronectin (Collins 1987).

In our study, fibrinogen adhesion on PC-coated PMMA was approximately half that

seen for the other test materials. An indication o f the possible importance o f this

reduction in fibrinogen binding is given by the results o f the fibrin-binding assay in

which adhesion was virtually eliminated by the PC coating. Fibrinous occlusion is a

common complication with all forms o f GFS. In clinical series, 8 - 11% o f GDDs are

occluded by fibrin in the early postoperative period (Chapter 1). Partial occlusion or

significant fibrinous surface spoilation may be considerably more prevalent.

Partial occlusion is o f particular relevance to GDDs in which small bore filtration

channels are used as a resistance mechanism. From Poiseuille's formula, typical

diameters required to regulate flow effectively at physiological aqueous outflow rates

lie in the range 20-60pm for single tubes up to 6mm in length (Chapter 2). At these

diameters, small accretions o f biological debris could lead to relatively large

percentage changes in cross-sectional area and flow performance.

In these studies, the PC-coating also reduced cellular adhesion for both macrophages

and scleral fibroblasts significantly. Differences between PC-coated PMMA and other

test materials were less dramatic than those observed in the protein adhesion assays.

This relative reduction in effect may again be attributable to protein conditioning. Pre­

coating with albumin passivates material surfaces (Kottke-Marchant 1989), reducing

subsequent adhesion o f other protein and cellular elements. Albumin in the culture

100

media used may have exerted a partial passivation effect in our assays reducing cell

adsorption to the un-coated materials. Differences between PC-coated and other test

materials were significant nonetheless.

Beyond surface spoliation, macrophage adhesion in particular is thought to be an

important initiator o f chronic inflammatory responses to implanted foreign material

(Tang 1995; Smetana 1997). Progressive fibrous encapsulation driven by a low-grade

foreign body inflammatory response limits the functional lifespan o f contemporary

GDDs (Table 5; Chapter 1; Page 27). The use o f materials which reduce fibroblast

and macrophage adhesion may significantly lengthen ODD filtration life.

The precise mechanism by which PC-polymers resist non-specific protein and cell

adhesion is not well understood. The natural cell wall phospholipid bilayer is self­

assembling. Stability is achieved by sequestering hydrophobic lipid moieties to the

interior in an aqueous environment. PC polymers may mimic natural cell surfaces by

preferentially adsorbing a self-assembling phospholipid monolayer in the correct

configuration (Ishihara 1992). Alternatively, resistance to protein and cell adhesion

may be mediated by properties intrinsic to the PC molecule (Hayward 1984, Durrani

1986). Phosphorylcholine is zwitterionic, possessing both positive and negative

electrical charges in close proximity, but is overall electrically neutral. The

zwitterionic nature o f the PC head group attracts a large hydration shell, and it is

possible that this relatively large and stable surrounding barrier o f water molecules

mediates resistance to protein adhesion

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3.7.3 Bovine Corneal Endothelial Damage

The amount o f endothelial damage caused by tube implantation, both at the time o f

operation and in association with subsequent micro-motion, probably varies with

physical force and the area o f contact. Where these factors are equal, as in our study,

the key determinant o f damage is the strength o f tissue to polymer adhesion. Comeal

endothelial cell damage caused by contact with silicone discs was less than that

caused by PM MA discs; but a PC polymer coating reduced endothelial damage to a

negligible level.

Standardised direct contact assays have previously used cadaveric animal corneas

(Kassar 1980, Barrett 1984, Mateo 1989). The use o f a cultured monolayer in this

assay has the advantage o f less inter-sample variation in association with pre-test

comeal condition and material to surface curvature mismatches. Although the strength

o f intercellular and cell to basement membrane adhesion in this culture model might

not perfectly mimic the conditions in-vivo, our findings for conventional materials are

consistent with those from previous studies in cadaver eyes (Kassar 1980, Barrett

1984, Mateo 1989).

Several factors are known to influence the strength o f cell adhesion to different

material surfaces. These include surface hydrophilicity (Ratner 1981), specific surface

chemistry (Moroudas 1973), surface rigidity (Yasuda 1978), and surface energy

(Kaufman 1977). Relatively hydrophobic surfaces such as PM MA were found to be

damaging to the endothelium in the nineteen seventies when the prevalence o f

pseudophakic bullous keratopathy began to increase (Knight 1979). Further in vitro

102

experimentation confirmed that hydrophilic surface modification o f PM M A or

substitution with more hydrophilic polymers could reduce endothelial damage in

standard touch tests (Barrett 1984, Mateo 1989, Balyeat 1989, Knight 1979).

Elastomeric silicone is also relatively hydrophobic, but has a lower surface free

energy than PMMA. Lower endothelial damage coefficients in both our own and

previous studies reflect this. Although less damaging than PMMA, the high rate o f

corneal endothelial failure after conventional tube implantation (Table 6; Chapter 1:

Page 29) suggests that the extent o f endothelial damage induced by contact with

silicone remains clinically significant.

PC polymers diminish contact induced comeal endothelial damage. This could help to

reduce the relatively high rate o f comeal endothelial failure associated with clinical

ODD implantation.

3.7.4 Bacterial Adhesion

The bacterial adhesion study demonstrated that modification o f PMMA discs with PC

polymer lead to a considerable reduction in the biomass o f adherent bacteria observed

in conditions o f standardised exposure to a clinical bacterial isolate. As with previous

studies (Raskin 1993, Dilly 1989), polypropylene showed a higher level o f bacterial

adhesion than the other test materials. An unusually high rate o f endophthalmitis has

been observed in association with intraocular lenses incorporating polypropylene

haptics (Bainbridge 1998).

Bacterial adhesion assays typically involve direct visualisation with scanning electron

or light microscopy (Raskin 1993, Dilly 1989), or radiolabelling o f bacteria (Raskin

103

1993). The bacterial biomass assay we used is objective, reproducible and devoid o f

radio hazard (Faragher 1996).

Many workers relate the adhesion o f bacteria to surfaces to the DLVO theory

(Derjaguin and Landau 1941, Verwey and Overbeek 1948) o f particle adhesion that

balances repulsive electrostatic interactions against attractive van der W aal’s

interactions between the surface and the organism (Garbassi 1998). Negatively

charged surfaces will have a tendency to repel bacteria which themselves possess a

negative charge (Dankert 1986). To some extent, this can be overcome by the ability

o f organisms to extend pili to provide an initial focal point o f attachment (Oga 1988).

In addition to charge, free surface energy and hydration may influence the ease o f

attachment. M aterials with a low surface energy, such as PTFE, are relatively inert

and resistant to attachment. Although most hydrogels have a relatively high free

surface energy, interfacial properties are modified by bound water (Tromp 1986). The

large hydration shell surrounding phosphorylcholine molecules in PC-PMMA may

explain the very low levels o f bacterial adhesion observed in this study.

PC-PMMA may diminish the risk o f endophthalmitis in ODD implantation.

3.8 Conclusion

Bioinert materials have been developed to improve the clinical performance o f

medical implants used in fluid contacting applications. To date, GDDs have been

constructed using conventional medical polymers. Our testing has demonstrated that a

104

highly promising new family o f bioinert polymers, PC polymers, out-perform these

conventional materials in each o f a panel o f in vitro assays designed to reflect

important dimensions o f biocompatibility in the context o f GDD implantation.

105

CHAPTER FOUR - Prototyping & In Vivo Study

Abstract

AIMS.

To assess the reproducibility o f the prototyping process for a novel glaucoma drainage

device (GDD) and the performance o f prototypes in vivo.

METHODS

Prototypes were machined from PMMA block, and micro drilling was carried out

using an excimer laser aiming to create a quasi-cylindrical flow resistance pathway

(average diameter 35 pm). The pressure drop across each prototype device was

measured on a custom flow rig, with the flow rate set at 2 pl/min. Prototype glaucoma

drainage devices, which produced the required pressure drop across the device (5-20

mmHg), were implanted in New Zealand White rabbits. lOPs were monitored using a

pressure transducer linked to the anterior chamber via a 27g cannula for 30 minutes

after the operation.

RESULTS

50 prototype GDDs were machined and laser drilled. All were coated with PC

polymers. 48 glaucoma drainage devices were rejected after the flow rig assessment.

The two remaining prototype GDDs were implanted without complications and

produced good flow control, with lOPs in the target range throughout the monitoring

period, and no evidence o f fibrinous occlusion.

106

CONCLUSIONS

Despite material homogeneity and controlled manufacturing conditions, laser drilling

for PMMA was insufficiently accurate over a 1mm depth to produce good flow

control in the majority o f prototype GDDs. Prototypes which passed preoperative

flow control tests also performed well in vivo, providing good protection from

hypotony. Alternative manufacturing techniques will be required to realise the clinical

potential for new GDDs.

107

4.1 Introduction

In vitro experimentation had shown that it was possible, in principle, to control the

dimensions o f a manufactured tubular lumen in a GDD accurately enough to provide

consistent protection from hypotony in the early period after GFS. Blank prototype

devices with a modified external profile also demonstrated good protection from

external leakage. Having demonstrated the promise o f PC polymers as bioinert

coatings for GDDs, we aimed to produce a batch o f prototype PC coated GDDs

incorporating a single channel flow restrictor and the novel external profile. We also

aimed to assess the correlation between pre-clinical flow testing and flow control after

implantation in an animal model.

Our original flow control experimentation (chapter 2) was based on ablation through

75pm thick polyimide sheets. At this thickness, a 15pm diameter ablation was found

to produce a flow pathway with the correct resistance characteristics, giving a

pressure drop across the device, which lay consistently within the desired target range

(5-15mmHg). A single macrophage could block GDDs in vivo if the flow pathway

were this narrow. Accordingly, a decision was taken to scale-up the diameter in

prototype construction, whilst maintaining flow resistance by lengthening the flow

pathway (Figure 7b; Chapter 2; Page 48). From the Poiseuille's formula, for a tube

with length o f 500 pm, a diameter o f 35pm should impart the correct flow resistance

at physiological aqueous production rates.

108

4.2 Materials & M ethods

4.2.1 Prototyping

Prototype GDDs were machined from PMMA (ICI, UK) by Pearson Matthews

Design Partnership (Kingston-on-Thames, UK) (Figure 13; Chapter 2: Page 57).

The GDDs were 4 mm in length with a tapering end and a Tune’ profile externally. A

large-bore hole (0.5 mm diameter) was drilled to within 0.5 mm o f the tapered end o f

the device. Excimer laser drilling was then carried out at Excitech Ltd (Oxford, UK)

to produce a small round hole with an average diameter o f 35 pm connecting the

leading end o f the device to the large-bore, zero resistance portion. Prototype GDDs

were then coated with a PC based polymer (Biocompatibles Ltd., UK) by immersion.

4.2.2 Flow Rig

The flow rig used was similar to that previously described for flow evaluation in

GDDs (Porter 1997; Prata 1995). The system consisted o f a pressure transducer

(Model P23ID, Gould Inc., California, USA), bridge amplifier (Model M LIIO, AD

Instruments, N ew South Wales, Australia) and recorder (Model ML200, AD

Instruments, New South Wales, Australia). Pressure changes were recorded using a

MacLab software (v3.5 AD Instruments, New South Wales, Australia) on a computer

(Performa 630, Apple Ltd., California, USA). A I ml hypodermic syringe (Fortuna,

Germany) was used with an adjustable electronic syringe pump (Model PHD 2000,

Harvard Apparatus, M assachusetts, USA) to control the flow rate. A three-way lock

109

was positioned between the water reservoir and the rest o f the system. Silicone tubing

(Silicone High Strength Tubing, Altec, Alton, UK) was used throughout. The free end

o f the tubing was sealed with silicone adhesive and left to dry. Once solidified, a

small slit incision was made through the sealed end with a standard M VR blade

(Visitech, UK) and prototype GDD implanted. No leakage around the GDD was

observed throughout the experiment. The transducer and the recording system were

calibrated using a water manometer daily prior to experimentation. Once the system

was filled with water, all gas bubbles were flushed out with the device submerged so

that flow through the device exit was not affected by surface tension generated at an

air interface (Figure 30). At this point, the three-way lock was turned to open the

system to atmospheric pressure and the pressure reading zeroed on the computer. The

three-way lock was then turned to obtain a closed system and infusion pump started.

The flow rate was set at 2 □ 1/minute. The pressure reading was allowed to stabilise

over 20 minutes, and the average reading from the final 10 minutes was then taken as

the pressure drop for the experiment. This recording was repeated six times for each

GDD. Devices producing a pressure drop o f 5-20mmHg were accepted for

implantation.

4.2.3 Animal model

Healthy adult New Zealand White rabbits were selected with weights between 2 and 3

kilograms. Baseline examinations were carried out and all eyes involved in the study

were normal. Throughout the experimentation, animals were cared for by experienced

animal handlers in accordance with the Helsinki Declaration and ARVO protocols for

the welfare o f experimental animals.

1 10

Figure 30. A schematic drawing of the How rig to test the prototype GDDs. The system consisted o f a pressure transducer (Model P23ID, Gould Inc., California. USA), bridge amplifier (Model M L llO . AD Instruments. New South Wales.Australia) and recorder (Model ML200. AD Instruments. New South Wales,Australia). Pressure changes were recorded using a MacLab software (v3.5 AD Instruments. New South Wales. Australia) on a computer (Performa 630. Apple Ltd., California. USA). A 1 ml hypodermic syringe (Fortuna. Germany) was used with an adjustable electronic syringe pump (Model PHD 2000, Harvard Apparatus,Massachusetts, USA) to control the flow rate. A three-way lock was positionedbetween the water reservoir and the rest o f the system. Silicone tubing (Silicone High Strength Tubing, Altec. Alton. UK) was used throughout. The free end o f the tubing was sealed with silicone adhesive and left to dry. Once solidified, a small slit incision was made through the sealed end with a standard MVR blade (Visitech. UK) and prototype GDD implanted.

W a t e r Rese rvo i r

T h re e -w ay Lock

Ma}>nifled View o f tube end

e on Infusion p u m p

Pres su reT r a n s d u c e r

G D D im p la n t ed at the sealed end

C om puter

I l l

4.2.4 Study Design

This was a prospective, internal, unmasked, efficacy study. Two rabbits were

implanted with GDDs shown to produce a cross-device pressure drop in the target

range (5-15mmHg) in preoperative testing. Each rabbit was implanted with the GDD

in one eye only under general anaesthesia. Implants were flushed with Balanced Salt

Solution (BSS). lOP was monitored continuously using a pressure transducer for 30

minutes after the operation with anterior chamber (AC) infused (F igure 31). The

rabbits were examined and assessed for TOP, AC depth/inflammation and bleb

morphology daily in the first week and then twice weekly for the next five weeks. On

day 42, the rabbits were sacrificed.

4.2.5 General Anaesthesia

The rabbit was held firmly by the shoulders and intramuscular ketamine (50 mg/Kg)

and xylazine (10 mg/Kg) given intramuscularly into the most muscular part o f the

thigh. A 5ml syringe was used with a disposable 23 gauge needle for the injection.

The anaesthetic was allowed to take effect and further procedures were carried out

when the rabbits were fully anaesthetised.

112

Figure 31. A schematic drawing o f the intraocular pressure monitoring set-up. The 27 gauge needle primed with balance salt solution was inserted into the anterior chamber and connected to a pressure transducer and computer to record the real time intraocular pressure changes.

W ater Reservoir O peratingM icroscope

Three-w ay Lock

27g needle inserted into AC

D

PressureTransducer

Computer

BP Am plifier

PowerLab 200

113

4.2.6 Implantation of Prototype Glaucoma Drainage Device

The anaesthetised rabbits were placed under the operating microscope with the

eyelids held open with a speculum. A fine 7/0 silk traction suture was placed through

the superficial limbal tissues to pull the eye down. A 27 gauge butterfly needle (filled

with BSS) connected to a digital pressure transducer was used to monitor the lOP

changes (Figure 31). The 27 gauge needle was carefully introduced through a comeal

stab incision parallel to and well anterior to iris plane. A fomix based conjunctival

flap was fashioned, and a selerocorneal slit incision was made parallel to the iris

plane using a standard 1.19mm width mierovitreoretinal (MVR) blade to enter the

anterior chamber. The GDD was inserted through the wound, and sutured with 10/0

nylon (Figures 32-33). The conjunctiva was sutured with 10/0 vicryl. A sub­

conjunctival injection o f betnesol and cefuroxime was administered at the end o f the

procedure. lOP was monitored continuously for over 30 minutes before the removal

o f the 27-gauge needle from the anterior chamber and conjunctival closure.

1 14

Figure 32. A schematic drawing o f the glaucoma drainage deviee in-si In. Fornix based conjunctival flap was fashioned, and a selerocorneal slit incision was made parallel to the iris plane using a standard 1.19mm width mierovitreoretinal (MVR) blade to enter the anterior chamber. The glaucoma drainage device was inserted through the wound, and sutured with 10/0 nylon, dhe conjunctiva was sutured with 10/0 vicrvl.

Conjunctiva

Cornea

Sclera

Pro to ty p e g l a uc om a d ra i n a g e device

15

Figure 33. Prototype glaucoma drainage device in situ. Week 2 post operation. The tip of the device (A) is just visible in the anterior chamber. The external part of the device (B) is sutured with 10.0 nylon and covered by conjunctiva.

] 16

4.2.7 Follow-up Examination

Eyes were examined under topical anaesthesia using a portable slit-lamp. The anterior

chamber depth, the degree o f inflammation and the bleb morphology were graded and

recorded. Eyes were then photographed with a standard 35mm camera. lOP was

measured using a Tonopen. Examinations were repeated daily for the first week, and

twice weekly for the next 5 weeks to monitor for the following adverse events; AC

fibrinous reaction, systemic illness, com eal abrasion, corneal oedema, hypopyon and

device extrusion.

117

4.2.8 Sacrifice

Animals were sacrificed using a lethal intravenous injection o f phenobarbitone under

general anaesthesia.

4.3 Results

4.3.1 Pre-operative flow testing

O f the 50 prototype GDDs that were machined, laser drilled and coated with PC

polymers, only 2 prototype GDDs fulfilled the pressure drop requirements after flow

rig assessment.

4.3.2 Adverse Reactions

The anterior chamber remained well formed through out the study period, and no

fibrinous reaction was observed in the anterior chamber. No systemic adverse reaction

was observed at any stage post implantation.

4.3.3 Intraocular Pressure

Rabbit 1

The implanted GDD had a cross-device pressure drop measured at 19 mmHg, at a

flow rate o f 2 □ 1/minute and the baseline IGF in the right and left eye were 19 mmHg

118

and 21 mmHg respectively. For the fist 30 minutes after implantation, the implanted

eye’s TOP maintained a pressure o f between 19 mmHg and 21 mmHg. Controlled

fluid escape through the GDD was visible throughout the monitoring period. There

was no anterior chamber shallowing, or evidence o f external leakage. The tonometric

10? of both eyes during the period o f the study is shown in Figure 34. The lO P’s in

the operated eye were lower than the control eye in most measurement until the third

week and no bleb was visible after day 8 (the error o f lOP measurement in rabbits

using Tonopen is ± 3 mmHg).

Figure 34. The intraocular pressures o f both eyes o f rabbit 1 during the study.

lO P in m m H g

22

20

18 -

16

14

12

Control Eye GDD Eye

10 20 30 40

Post-O perative D ay

50

119

Rabbit 2

The implanted GDD has a pressure gradient o f 6 mmHg at a flow rate o f 2 □ 1/minute

and the baseline lOP in the right and left eyes were 21 mmHg and 20 mmHg

respectively. For the first 30 minutes after implantation, I OP was maintained at 17-18

mmHg. Again, controlled flow was visible through the GDD, and no anterior chamber

shallowing was observed. The lOP o f both eyes during the follow up period is shown

in Figure 35. The lO P’s in the operated eye were lower than the control eye in most

measurement until the second week and no bleb was visible after day 8.

Figure 35. The intraocular pressures o f both eyes o f rabbit 2 during the study.

lO P in m m H g

28

22

Control Eye GDD Eye

10 20 30 40 50

Post-O perative D ay

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4.4 Discussion

Using current machining and the excimer laser techniques, critical dimensional

control was too poor to provide a reproducible internal flow resistance in the desired

target range (5-20mmHg for this phase o f the study). In contrast to earlier studies

(chapter 2) in which excimer laser ablation o f 75 pm thickness polyimide sheeting was

found to produce a high level o f dimensional reproducibility, and flow control within

a finer target range (5-15mmHg); we had selected a longer (500pm), larger-bore

(35 pm) resistance channel for this set o f prototypes, and chosen PMMA as a

manufacturing material. The choice o f PMMA was based on ease o f PC-polymer

coating, and a long track record o f safety for this material in the eye. As explained

above, a larger-bore resistance channel was felt to be necessary in vivo to minimise

the chances o f blockage with a single cell or pre-formed accretions o f biological

debris. Both the change in material and the change in dimensions may have

contributed to the inaccuracy o f ablation. Polyimide is superior to PM MA as an

ablation substrate, and the high aspect ratio o f the longer resistance channel would

tend to lead to variable masking effects caused by inadequate exhaust o f ablation

products between pulses.

Although the poor yield from prototyping only afforded a very preliminary view o f

the clinical potential for the new GDDs, the devices that were implanted performed

well. The tapered front section facilitated implantation with minimal tissue

manipulation, and implantation for the lune-shaped prototypes through a slit incision

was a simple process compared to that for existing GDDs. Selected prototype GDDs

121

good flow control with no hypotony or shallowing o f the anterior chamber. PC-

coating o f PMMA has no notable adverse systemic or local reaction in rabbits.

Notably for the rabbit model, there was no evidence o f fibrinous occlusion, or any rise

in lOP, which might suggest partial occlusion during the immediate post implantation

observation phase.

4.5 Conclusion

Although these studies fall some way short o f securing proof o f principle for the

design concepts developed in vitro, they do provide a tantalising glimpse o f the

possibilities for a new generation o f GDDs in vivo. Further exploration o f micro­

manufacturing techniques required to produce the requisite level o f critical

dimensional control will be necessary before this potential can be realised in clinical

trials.

122

CHAPTER FIVE- Concluding Discussion & Future Development

5.1 Discussion

The premises underpinning the rationale for using a novel GDD in GFS are: first, that

consistent internal flow control and elimination o f external leakage will prevent early

post-operative hypotony; and second, that using a bioinert coating will minimise

modification o f internal flow resistance by biological spoliation, and minimise any

foreign body inflammatory reaction.

Experimental flow studies described in preceding chapters demonstrate that

Poiseuille’s law will accurately predict the critical dimensions for a GDD flow

restrictor based on a small bore tube. They also demonstrated that improving fit

between the external profile o f a new GDD and the incision through which it is

inserted should facilitate implantation and eliminate uncontrolled external leakage.

Our in vitro assays, designed to reflect a number o f key dimensions o f

biocompatibility in GDD implantation, suggesting that new generation bioinert

materials should improve clinical performance. Further information could be obtained

from sub-conjunctival implantation studies o f material biocompatibility and dynamic

flow performance in conditions reflecting the aqueous environment after blood

aqueous barrier breakdown using various concentrations o f serum in the flow rig.

Although limited, our initial observations o f flow performance in vivo were highly

encouraging. There were no adverse tissue reactions post-operatively. Overall, work

contained in this thesis demonstrates proof o f principle for a number o f important new

design departures in GDD development.

With current laser drilling techniques, dimensional control is too poor to make bulk

manufacture o f GDDs, or even prototype manufacture for further trials, viable. The

challenge now is to identify a more reliable means o f critical dimensional control.

123

5.2 Future Development

5.2.1 Extrusion M oulding and Hot Drawing

Extrusion is a process that can be compared to squeezing toothpaste out o f a tube.

Polymer granules are forced through a heated barrel and the fused polymer is then

squeezed through a die, which defines the profile o f the extruded component. Water

or air-cools the extruded material as it leaves the die, and is cut to the required length.

Disadvantages are that the cost o f producing a die is high and the process is limited to

shapes with a uniform cross-section. This would limit design flexibility for a new

device.

Hot drawing is similar to extrusion, but involves heating a thermoplastic polymer or

glass to its glass transition temperature, then exploiting the consistency o f length to

diameter ratios produced by stretching a heated tubular element under constant

tension. This is the process by which glass micropipettes in routine laboratory use are

fabricated. Hot drawing is also limited to a uniform cross-section; but is relatively

cheap to explore, and could be a viable route to producing a limited prototype run.

5.2.2 Photolithography

Photolithography is a relatively new micromanufacturing technique in which

polymers are exposed to selectively to light energy in order to define a three

dimensional shape. Selective exposure is either through a mask to produce a uniform

cross-sectional shape, or by scanning a focussed point o f light through the target

polymer, or “photoresist,” to produce non-uniform cross-sections. Light energy

124

defines shapes within the resist either by curing the polymer (positive photoresists) or

ablating it (negative photoresists) at the point o f exposure. Shorter wavelength light

defines shapes most accurately, and X-ray lithography is capable o f producing

complex shapes with submicron accuracy. These accurate solid representations o f

designs on a micron scale can be used either to create prototype devices individually,

or to create micro-moulds for scale up to bulk manufacture.

5.3 Conclusion

The health economic arguments for developing a fast, reliable, one-stop treatment

glaucoma are clear, particularly in the developing world, where access to regular

review and drug treatment is likely to remain limited. Laser drilling has not been

accurate enough to realise the goal o f producing a biocompatible GDD to improve

flow control and reduce operating time in routine GFS. Emerging techniques for

micro-manufacture such as X-ray lithography hold immense promise in this field.

Possibilities for fusing these techniques with improvements in material

biocompatibility and advances in the pharmacological control o f wound healing

suggest a bright future for GDDs in the new century.

125

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A ppendix 1

Glaueoma valves: Truth versus myth II

Dear Editor:

Lee' has recently exposed the theoretical flaws in the designs o f both the Ahmed and

Krupin Valves, leading marketed glaucoma drainage devices (GDD). Theoretical

flaws may also exist for the OptiMed GDD (Model 1014, Tecfen Corporation, Santa

Barbara, California, USA).

The flow resistance mechanism for the OptiMed device consists o f a small block o f

parallel tubules. No quoted diameter or range o f diameters is either given on the

company web site (www.tecfen.com/medical/ophthal/optimed.html) or published.

We examined a sample OptiMed device, using a field emission scanning electron

microscope (JEOL, Akishima, Japan) and a image analysis software (Scion Corp.,

Maryland, USA). 392 tubules were identified in a PMMA block measuring 3x2.2x0.7

mm. The average diameter o f the tubules was 47.5pm (Figure 1). With a series o f

tubules in parallel, the pressure drop across the device would vary in accordance with

a modification o f Poiseuille's formula^: -

Pressure drop = 128plQ / \36nd"^ N

Where

n = aqueous viscosity I = length o f tube Q = aqueous flow rate d = diameter o f tube N = Number o f tubes in parallel

When applied to glaucoma drainage device, the average aqueous flow rate (Q) can be

assumed" to be 2 pl/min with a viscosity (p) o f 0.001 Nsec/m^. This gives a pressure

drop across the sample device o f only 0.01 mmHg. Even in a patient with an aqueous

flow rate o f 3.1 pl/min% and aqueous viscosity ten times that o f normal, the pressure

drop across the device would still only be 0.169 mmHg.

Recent flow studies'*^ have shown that OptiMed devices did not function as claimed,

this is not surprising in view o f the simple flow analysis given above. No clinical

140

results have been published. Even in the absence o f leakage around the device, a

common problem in GDD implantation, it seems unlikely that the OptiMed device

would offer effective protection from early post-operative hypotony.

References

1. Lee VW. Glaucoma “valves” - truth versus myth, [letter] Ophthalmology

1998;105(4):567-8

2. McEwen WK. Application o f Poiseuille’s law to aqueous outflow. Archives o f

Ophthalmology 1958;60:290-294

3. Brubaker RE. Flow o f aqueous-humor in humans. The Friedenwald Lecture.

Investigative Ophthalmology & Visual Sciencel991;32(13):3145-3166

4. Prata JA, Mermoud A, LaBree L, Minckler DS. In vitro and in vivo flow

characteristics o f glaucoma drainage implants. Ophthalmology 1995; 102: 894-

904

5. Francis BA, Cortes A, Chen J, Alvarado JA. Characteristics o f glaucoma drainage

implants during dynamic and steady-state flow conditions. Ophthalmology

1998;105:1708-1714

141

Figure 1. Scanning electron microscopy image o f a sample Optimed device How restrictor.

^ A.4S G ^

t» .

142

Appendix 2

Rabbit Study Protocol Forms

143

Cage Label

AGFID ANIMAL Study 1

Adyerse Reaction Form Date: / /

Eye

Date o f Operation; Days Post-Op:___

OD/OS

/ /

lOP: OD

Complications:

Systemic illness

Comeal Abrasion

Corneal Oedema

Hypopyon

Deyice Extrusion

Others:

mmHg OS mmHg

Yes No Comments

TREATMENTS

OUTCOME

Inyestigator Signature Date Co-ordinator Signature

144

Cage Label

AGFID ANIMAL Study 1

Operation Records Date: / /

Eye OD/OSAGFID Device Lot #:

Operator:Assistant:Time: Starting Completion

MMC: Concentration Time Area

AC Reform: □ Yes □ No

Drues injected into AC

Complications: Yes No

Excessive sub-conjunctival haem orrhaee [ 1 nConjuctival damaee n nHvnhaema n nPoor anaesthesia n nOthers

Comments:

Investigator Signature Date Co-ordinator Signature

145

Cage Label

AGFID ANIM AL Study 1

Post-operative 10? monitoring study Date: / /

Eye GD/GS

Lot #

IO P:Pre-opGD GS

Post-op

IGF Monitoring

Device: AGFID/Standard

Agent injected into AC DHeparin DTP A DNone

Position of AC tube: DVisible DNot Visible

Cornea:______ DCiear______________ DHazv DVerv Hazv

AC Depth: DFlat DShallow DFormed

Hyphaema: 0100% O>50% D>25% 0<25% GNone

Pre-Clamping IGF: mmHg

Maximum IGF: ____ mmHg

Time Taken to Reach:____ Minutes

Comments:

Investigator Signature Date Co-ordinator Signature

146

Cage Label

AGFID ANIMAL Study 1

Post-Operative Assessment Date: / /

Slit Lamp ExaminationOperated Eye OD/OS

Date o f Operation: / /Davs Post-Op:

IGF: OD mmHg OS mmHg

AC Depth: DFlat

AC Inflammation: □++++

□Shallow DFormed

D+++ D++ D+ D-

Bleb Appearance: DVascular DAvascularHeight_____Area

Comments:

Investigator Signature Date Co-ordinator Signature