GOSS, Ben (44 Jones Road, Carina, Queensland 4152, AU)
LUTTON, Cameron (62 Clarendon Street, East Brisbane, Queensland 4169, AU)
GOSS, Ben (44 Jones Road, Carina, Queensland 4152, AU)
CLAIMS
1. A biocompatible polymeric material for tissue repair comprising at least one negatively charged monomer and at least one charge neutral co-monomer, wherein the biocompatible polymeric material induces thrombogenesis and reduces a complement-mediated foreign body response.
2. The biocompatible polymeric material of claim 1, wherein the at least one negatively charged monomer is selected from the group consisting of acrylic acid,
di-2-propenyl-acetic acid, crotonic acid, vinylacetic acid, methacrylic acid, propylenedicarboxylic acid, 2-propene-l -sulfonic acid, vinyl sulfonic acid, vinyl
acetic acid, butenoic acid and propenoic acid.
3. The biocompatible polymeric material of claim 2, wherein the at least one
negatively charged monomer is acrylic acid.
4. The biocompatible polymeric material of claim 1, wherein the at least one charge neutral co-monomer is selected from the group consisting of methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, ethylene glycol diacrylate, hydroxyethyl methacrylate, di-
methacrylated sebacic acid anhydride, di-methacrylated adipic acid anhydride, caprolactone, lactone, glycolide, dimethacrylated adipic acid anhydride and
dimethacrylated sebacic acid anhydride. 5. The biocompatible polymeric material of claim 4, wherein the at least one
charge neutral co-monomer is selected from the group consisting of methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl methacrylate, di-methacrylated adipic acid anyhydride and dimethacrylated sebacic acid anhydride.
6. The biocompatible polymeric material of claim 1, wherein the at least one negatively charged monomer is at a concentration of between about 15% and 85%.
7. The biocompatible polymeric material of claim 6, wherein the at least one negatively charged monomer is at a concentration of between about 45% and 65%.
8. The biocompatible polymeric material of claim 1, wherein the at least one charge neutral co-monomer is at a concentration of between about 15% and 85%.
9. The biocompatible polymeric material of claim 1 , wherein the biocompatible polymeric material is suitable for tissue repair in an animal.
10. The biocompatible polymeric material of claim 9, wherein the animal is a
mammal. 11. The biocompatible polymeric material of claim 10, wherein the mammal is a human.
12. A biocompatible device for tissue repair comprising the biocompatible polymeric material of any of the preceding claims.
13. The biocompatible device of claim 12, wherein the biocompatible device is
selected from the group consisting of a bone graft, an implant and a wound dressing.
14. A method for synthesising a biocompatible polymeric material for tissue repair that induces thrombogenesis and reduces a foreign body response, said method
including the step of combining at least one negatively charged monomer and at least
one neutral co-monomer to thereby generate said biocompatible polymeric material
for tissue repair that induces thrombogenesis and reduces a complement-mediated
foreign body response.
15. The method of claim 14, wherein the at least one negatively charged monomer is selected from the group consisting of acrylic acid, di-2-propenyl-acetic acid,
crotonic acid, vinylacetic acid, methacrylic acid, propylenedicarboxylic acid, 2- propene-1 -sulfonic acid, vinyl sulfonic acid, vinyl acetic acid, butenoic acid and
propenoic acid.
16. The method of claim 15 , wherein the at least one negatively charged monomer
is acrylic acid. 17. The method of claim 14, wherein the at least one charge neutral co-monomer is selected from the group consisting of methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, ethylene glycol diacrylate, hydroxyethyl methacrylate, di-methacrylated sebacic acid anhydride, di-methacrylated adipic acid anhydride, caprolactone, lactone, glycolide, dimethacrylated adipic acid anhydride and dimethacrylated sebacic acid anhydride.
18. The method of claim 17 , wherein the at least one charge neutral co-monomer is selected from the group consisting of methyl methacrylate, methyl acrylate, ethyl
acrylate, ethyl methacrylate, di-methacrylated adipic acid anyhydride and dimethacrylated sebacic acid anhydride. 19. A biocompatible polymeric material for tissue repair produced according to the
method of claim 14. |
TITLE A PROTHROMBOGENIC BIOMATERIAL FOR TISSUE REPAIR
FIELD OF THE INVENTION
The present invention relates to a biomaterial. More particularly, the present invention relates to a biomaterial for use in tissue repair.
BACKGROUND TO THE INVENTION
Wound healing of many tissues is traditionally broken down into three main stages: the inflammatory phase, proliferative phase and remodelling phase. These
phases overlap both temporally and spatially as such the success of the preceding phase determines the effect of the subsequent phase. The initial response of the body to injury and as such the only phase of healing without the influence of preceding phases is to form a blood clot. This step has many physiological functions. Initially its role is the achievement of haemostasis and subsequent prevention death from
blood loss. The activation of platelets on a surface induces the release of tissue factor
(factor III), adenosine-5' -diphosphate (ADP), Thromboxane (Tx), Serotonin, and
other coagulation factors activating the extrinsic and propagating the intrinsic
coagulation pathways. This initial platelet plug reduces the shear stress between the platelets and the surface to enable the direct attachment of the platelets to damaged
tissue. The acceleration of the coagulation reaction occurs on the surface of the activated platelets via a prothrombinase complex composed of calcium, proteins, and the platelet phospholipids, phosphatidylinositol and phosphatidylserine. Normally these negatively charged lipids are not present on the outer cell membrane in sufficient concentrations to support effective initiation or propagation of the
coagulation responses. The intrinsic and extrinsic systems converge at factor X to a
single common pathway responsible for the activation of prothrombin to thrombin and in turn activation of the fibrin peptides A and B. These peptides polymerise into a three dimensional network and which stabilises the clot by the crosslinking action of factor XIII, achieving haemostasis.
Haemostasis is not the only function of the blood clot. The clot provides both
growth factors a cytokines to signal cell migration and proliferation as well as providing a scaffold for the invading cells. The clot extends across the wound and is
the first tissue to connect the ends of the edges of the wound. In a non-critical defect
macrophage infiltration occurs within 48 hours, which gradually removes the nonviable material. As conditions in the clot become hypoxic the macrophages platelets and red blood cells release a battery of angiogenic growth factors including tumour
necrosis factor (TNFα), interleukins 1 and 6, transforming growth factor α and β,
insulin-like growth factor (IGF) and platelet derived growth factor (PDGF). If the clot
remains stable it takes only a few days before capillaries begin to grow in. Fibroblasts migrate either from the peripheral tissue into the clot and produce a collagen matrix
known as granulation tissue.
The early migration of fibroblasts and myofibroblasts into the wound site
during this phase results in the release of a battery of pro-migratory and angiogenic
growth factors. Transforming Growth Factor β (TGFβ) 1 , Epidermal Growth Factor
(EGF) 2 , Fibroblast Growth Factor (FGF) 3 , Vascular Endothelial Growth Factor (VEGF) 4 , Platelet Derived Growth Factor (PDGF) 4 and Insulin Like Growth Factor
(IGF) 5 and a raft of other inflammatory cytokines including Interleukin-1 (ILl) 6 and
Interferon γ (IFNγ) 4 , which are all released in a sustained fashion for several days.
The fibroblasts and myofibroblasts produce a complex extra cellular matrix (ECM) that replaces the initial fibrin clot. This ECM consists primarily of collagen I, III, IV,
VIII, proteoglycans and glycoproteins including fibronectin 7 . Effectively the role of this tissue is to provide the signals and the structure for the migration, proliferation and differentiation of cells leading to the formation of the mature tissue type. The formation of a stable, vascularised granulation bed supports the influx of progenitor cells. These cells are differentiated by a series of environmental cues and begin to lay down extracellular matrix typical of their phenotype. Both the blood vessels and the tissue begin to mature and the remodelling phase of wound healing begins.
The formation of the blood clot is the first and only independent event that occurs during wound healing, all others are dependant to some extent on the
preceding stage. The mechanism of the formation of these clots is directly related to
the ability of the wound to heal. Clots that do not form a stable bridge across the wound, either as a result of impaired coagulation or large defects result in the failure of the wound to heal. Clots that are initiated through complement pathways induce massive foreign body response and prevent the occurrence of the subsequent stages.
The use of biomaterials in the body is aimed at bridging defects. Very little
attention is paid to the interaction of blood with these materials, inevitably leading impairment of coagulation on the material surface or activation of the complement
system. Artificial materials such as polymers, ceramics and allograft tissues in the
wound effects the initial attachment of platelets and complement proteins. The
competition for the surface between complement proteins and platelets is tipped in favour of complement proteins. These signal the up-regulation of the complement
cascade causing leukocyte activation and the release chemotractants to recruit
immune cells to the biomaterial interface. Immune cells adherent on biomaterial
surfaces produce a wide variety of active cytokines, including tumour necrosis factor-
α, and interleukins-1,6,8,10. These collectively mediate the foreign body response.
This chronic response both up-regulates inflammation and down-regulates wound healing 8 . Whilst a small clot still forms, the proportion of activated platelets in the clot is reduced and subsequent release of angiogenic factors is insufficient for neo-
vascularisation of the defect. Without the formation of the capillary network there can be no granulation tissue and no reduction in inflammation. This leads to the formation of a fibrous non-union or scar or encapsulation of the graft material. Despite technological advances in the development of biomaterials, there
exists a need for a biomaterial that evokes the same coagulation response as damaged
tissue.
SUMMARY QF THE INVENTION
In one form, although it need not be the only or indeed the broadest form, the
invention provides a biocompatible polymeric material for tissue repair comprising at least one negatively charged monomer and at least one charge neutral co-monomer
wherein the biocompatible polymeric material induces thrombogenesis and reduces a complement-mediated foreign body response.
Preferably, the biocompatible polymeric material induces thrombogenesis
whilst simultaneously reducing complement-mediated foreign body response.
In a second form, the invention provides a method for synthesising a biocompatible polymeric material for tissue repair that induces thrombogenesis and
reduces a foreign body response, said method including the step of combining at least
one negatively charged monomer and at least one charge neutral co-monomer to
thereby generate said biocompatible polymeric material for tissue repair that induces thrombogenesis and reduces a complement-mediated foreign body response. hi a preferred embodiment, the biocompatible polymeric material for tissue
repair comprises a surface that induces thrombogenesis and reduces complement- mediated foreign body response.
Preferably, the at least one negatively charged monomer comprises an acidic functional group.
More preferably, the at least one negatively charged monomer is selected from the group consisting of acrylic acid, di-2-propenyl-acetic acid, crotonic acid,
vinylacetic acid, methacrylic acid, propylenedicarboxylic acid, 2-propene- 1 -sulfonic acid, vinyl sulfonic acid, vinyl acetic acid, butenoic acid and propenoic acid.
Even more preferably, the at least one negatively charged monomer is acrylic acid.
Preferably, the at least one charge neutral co-monomer is selected from the group consisting of methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl
methacrylate, butyl methacrylate, isobutyl methacrylate, ethylene glycol diacrylate, hydroxyethyl methacrylate, di-methacrylated sebacic acid anhydride, di- methacrylated adipic acid anhydride, caprolactone, lactone, glycolide, dimethacrylated adipic acid anhydride and dimethacrylated sebacic acid anhydride. More preferably, the at least one charge neutral co-monomer is selected from
the group consisting of methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl methacrylate, di-methacrylated adipic acid anyhydride and dimethacrylated sebacic
acid anhydride.
Preferably, the at least one negatively charged monomer is in a concentration
range between about 15% and 85%.
More preferably, the at least one negatively charged monomer is in a concentration range between about 45% and 65%.
Suitably, said biocompatible polymeric material is biodegradable or non- biodegradable.
Throughout this specification, unless the context requires otherwise, the
words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
BRIEF DESCRIPTION OF THE FIGURES
In order that the invention may be readily understood and put into practical
effect, preferred embodiments will now be described by way of example with reference to the accompanying figures wherein like reference numerals refer to like parts and wherein:
FIG 1 is a X-ray photoelectron spectra (XPS) of a series of methyl
methacrylate-co-acrylic acid copolymers (MMA:AA:).
FIG 2 is the relative surface acid concentration measured by XPS for different copolymer formulations.
FIG 3 is a scanning electron micrograph showing the attachment of whole blood to a MMA55%:AA45%: showing the formation of a fibrin and platelet rich
coagulation plug and subsequent coagulation behaviour in the presence of the
surface.
FIG 4 is a scanning electron micrograph showing a top down view showing
the attachment of whole blood to a glass surface .
FIG 5 is a histological section of a blood clot formed on a MMA35%:65%AA surface, stained with DAPI for nuclei / leukocytes
FIG 6 is a histological section of a blood clot formed on a glass surface, stained with DAPI for nuclei / leukocytes FIG 7 is a graph showing leukocyte counts per 0.25mm 2 on the surface of
MMA35%:65%AA, MMA45%:55%AA, MMA55%:55%AA and glass.
FIG 8 is a graph showing concentration of serum anaphylotoxin (C5a) collected from heparinised whole blood samples incubated with the surfaces for 2
hours. FIG 9 is comparative, reproducible histological section of a blood clots formed on a MMA35%:65%AA surface, stained with DAPI for nuclei / leukocytes
from donors b-19 and 1-3.
DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated, in part, on the development of a biocompatible polymeric material suitable for tissue repair which is substantially
improved over conventional biomaterials. Throughout this specification, the term
"biomaterial" is used to refer to a material which is able to replace and/or treat natural body tissue. Such a biomaterial may be either synthetic or natural in origin
and may be biodegradable, depending on the application for which it will be used. The term "biocompatible " is used to refer to the ability of a biomaterial to
perform with an appropriate host response in a specific application. The host may be a mammal such as, but not limited to, humans, domestic pets, livestock and
performance animals.
In particular, the biomaterial of the present invention provides a superior
synthetic tissue substitute which has the effect of initiating wound healing by
promoting thrombogenesis, whilst simultaneously suppressing the foreign body immune response of the host. The ability to down-regulate the host immune response is of particular importance when the biomaterial is used for applications such as bone
grafts, wound dressings and/or implants.
Therefore in one broad form, the invention provides a biocompatible polymeric material for tissue repair that induces thrombogenesis whilst minimising a
host complement-mediated foreign body response.
The present invention provides a substantial departure from conventional approaches to biomaterial development. Typically, conventional biomaterials initiate
a complement-mediated foreign body response leading to local environmental changes that are not conducive to wound healing or are bio-inert through the strong denaturing actions of their surface. The biocompatible polymeric material composition of the present invention utilises a unique surface chemistry to promote, inter alia, tissue repair and regeneration.
There is no obvious relationship between surface chemistry and coagulation
or complement response. This is exemplified by the ongoing search for a truly haemocompatible material. Although not wishing to be bound by any particular
theory, the present invention demonstrates that both coagulation and complement
response are dictated by a combination of surface properties.
Upon blood-biomaterial contact there is an instantaneous adsorption of
proteins. The protein pattern of this adsorption depends on the surface chemistry of the material, and this event decides the outcome of the contact between blood and biomaterial. Central to this response is the competition for the surface between the
complement system and the attachment and activation of platelets and subsequent
induction of coagulation factors such as tissue factor (factor III), adenosine-5'- diphosphate, thromboxane, serotonin and others.
In the presence of conventional biomaterials, this initial platelet activation may not occur at all or may be hampered by the host immune response to a foreign body. Generally, the foreign body inflammatory response to a biomaterial is initiated by protein adsorption to that material surface followed by activation of the complement pathway to produce chemotactants for recruitment and activation of circulating leukocytes, which comprises a combination of neutrophils, monocytes, lymphocytes, basophils and eosinophils. The activation of leukocytes results in
release of inflammatory mediators such as elastase, cathepsin G and lactoferrin and
cytokines such as tumour necrosis factor α, interleukins 1, 6 and 8 and the like.
Often complement adhesion to a biomaterial surface is favoured over platelet adhesion. Therefore, it is highly desirable to modulate biomaterial-induced C3, IgG,
mannose binding lectin (MBL) adhesion and encourage platelet recruitment and attachment to the biomaterial surface. The present invention provides a solution to the propensity for attachment of these complement proteins over platelet adhesion to
a biomaterial by introducing a unique surface chemistry which favours platelet attachment and overwhelms or regulates complement-mediated foreign body
response.
Although not wishing to be bound by any particular theory, the present
inventors have determined that a suitable surface chemistry of a biomaterial is crucial
for the attachment of plasma proteins, platelets, up regulation of the coagulation cascade and down regulation of the complement response and hence subsequent
tissue repair cascade response.
In one aspect, the invention provides a biocompatible polymeric material for
tissue repair in which the biocompatible polymeric material comprises at least one
negatively charged monomer and at least one charge neutral co-monomer, wherein the biocompatible polymeric material induces thrombogenesis and reduces a complement-mediated foreign body response.
In the context of the present invention, by "induces thrombogenesis and reduces a complement-mediated foreign body response " is meant a biomaterial which has the ability to promote coagulation and the thrombogenesis cascade and is also able to reduce, diminish, control or minimise the complement-mediated foreign body immune response in comparison to a non-biocompatible polymeric material
which is not suitable for tissue repair. A complement-mediated foreign body immune response includes leukocyte activation by activation of the complement system. It is
contemplated that the reduction of the foreign body response may be concomitant with induction of thrombogenesis, although is not limited thereto.
In preferred embodiments, the biocompatible polymeric material includes a surface that induces thrombogenesis and reduces a complement-mediated foreign
body response.
In more preferred embodiments, the surface is a negatively charged surface. Preferably, the negatively charged surface initiates and promotes platelet
attachment and activation over complement protein attachment.
In particular preferred embodiments, the biocompatible polymeric material
comprises at least one monomer with a net negative charge. Non-limiting examples
of suitable net negatively charged monomers include acrylic acid, di-2-propenyl-
acetic acid, crotonic acid, vinylacetic acid, methacrylic acid, propylenedicarboxylic
acid, 2-propene-l -sulfonic acid, vinyl sulfonic acid, vinyl acetic acid, butenoic acid
and propenoic acid.
In preferred embodiments, the at least one negatively charged monomer is acrylic acid.
The biocompatible polymeric material further comprises at least one charge neutral co-monomer. Non-limiting examples of suitable co-monomers which are charge neutral include methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, ethylene glycol diacrylate, hydroxyethyl methacrylate, di-methacrylated sebacic acid anhydride, di- methacrylated adipic acid anhydride, caprolactone, lactone and glycolide.
In preferred embodiments, the at least one charge neutral co-monomer is selected from the group consisting of methyl methacrylate, methyl acrylate, ethyl
acrylate, ethyl methacrylate, di-methacrylated adipic acid anyhydride and dimethacrylated sebacic acid anhydride.
In particularly preferred embodiments, the biocompatible polymeric material comprises at least one negatively charged monomer in a concentration range between about 15% and 85%. It will be readily appreciated that in these embodiments, the remainder of the surface charge of the biocompatible polymeric material is neutral, wherein the at least one charge neutral co-monomer is in a concentration range
between about 15% and 85%.
In more preferred embodiments, the at least one negatively charged monomer
is in a concentration range between about 45% and 65%.
It is readily appreciated that the distribution of negative charges on the surface
of the biocompatible polymeric material may be either evenly distributed or alternatively, the negative charges may be concentrated in localised regions.
Suitably, said biocompatible polymeric material of the present invention may be biodegradable or non-biodegradable. A person skilled in the art will appreciate
that biodegradability may be imparted to the biomaterial of the invention by inclusion of at least one biodegradable co-monomer. Non-limiting examples of suitable
biodegradable co-monomers include di-methacrylated adipic acid anhydride and di- methacrylated sebacic acid anhydride.
In another general aspect, the invention provides a method for synthesising a biocompatible polymeric material for tissue repair that induces thrombogenesis and reduces a complement-mediated foreign body response, said method including the step of combining at least one negatively charged monomer and at least one charge neutral co-monomer to thereby generate said biocompatible polymeric material for tissue repair that induces thrombogenesis and reduces a complement-mediated
foreign body response.
In preferred embodiments, the method of the present invention includes
combining monomers with acidic functional groups and charge neutral co-monomers.
The biocompatible polymeric material of the present invention may be
generated in a number of ways, as are well known in the art. By way of example only,
the negatively charged surface may be generated by polymerising at least two monomers wherein each monomer comprises at least one free radically polymerisable
group. Examples of a suitable polymerisable group includes an ethylenically
unsaturated group, an unsaturated monocarboxylic acid, an allyl group, an unsaturated dicarboxylic acid, an unsaturated tricarboxylic acid, an acrylate and a
methacrylate, but are not limited thereto.
Alternatively, the negatively charged surface on the biocompatible polymeric material is generated by selectively modifying a functional group on a surface of a
biocompatible polymeric material by reactive species but is not limited thereto. It is also envisaged that the properties of the biocompatible polymeric
material surface can be manipulated by incorporation of other functional groups with particular physicochemical characteristics in order to influence biocompatibility. For instance, the biocompatible polymeric material may include a monomer with a
hydrophobic functional group to further aid platelet adhesion and activation. In light of the foregoing, it be appreciated that the biocompatible polymeric material of the invention may be synthesised from any combination of monomer, pre-
polymers or polymers that are polymerisable.
Therefore, one broad application of the present invention is a three- dimensional biomaterial with a unique surface chemistry that induces platelet activation in vivo whilst simultaneously minimising a host complement mediated
foreign body response. It is readily contemplated that by manipulation of the type and amount of monomers, such a material may be adapted to a wide variety of purposes
where a tissue defect has been incurred and a surface can be applied thereto to treat
the defect. By way of example only, polymerising methyl methacrylate, acrylic acid
and methacrylated sebacic anhydride in about a 30:65:5 ratio will produce a stiff biodegradable composition for use in applications which demand a rigid properties such as a bone graft whereas acrylic acid may be polymerised around a polyethylene
glycol/polylactic acid copolymer to form a flexible interpenetrating network material for skin or bandage applications. Therefore a particular advantage conferred by the
biocompatible polymeric material composition of the present invention is the amenability to production of biomaterials with wide a wide range of different properties such as degradability, non-degrading, rigid, flexible, high load bearing
capacity but is not limited thereto. These materials can be readily polymerised into various shapes for various applications for example beads for impactable bone grafts, sheets for wound dressings, or polymerised to the surface of an implant to produce bioactive implant coatings.
It will be appreciated that the biocompatible polymeric material is suitable for
tissue repair in an animal. An animal can be selected from the group consisting of humans, domestic livestock, laboratory animals, companion animals and
performance animals, although without limitation thereto. Preferably, the animal is a mammal. More preferably, the mammal is a human.
So that the present invention may be more readily understood and put into
practical effect, the skilled person is referred to the following non-limiting examples.
EXAMPLES
Materials and Methods
Preparation and polymerisation of Methyl methacrylate-co- acrylic acid (35:65)
A mixture of 35mol% methyl methacrylate and 65mol% acrylic acid was prepared. Benzoyl peroxide (0.5% w/w) was added. The resultant mixture was
polymerised at 65 0 C for 1 hour in an inert atmosphere. The polymer was post-cured
at HO 0 C for 15 minutes in an inert atmosphere, to produce a clear smooth polymer.
Preparation and polymerisation of Ethyl methacrylate-co- acrylic acid (35:65)
A mixture of 35mol% ethyl methacrylate and 65mol% acrylic acid was
prepared. Benzoyl peroxide (0.5% w/w) was added. The resultant mixture was polymerised at 65 0 C for 1 hour in an inert atmosphere. The polymer was post-cured
at HO 0 C for 15 minutes in an inert atmosphere, to produce a clear smooth polymer.
Preparation and polymerisation of Methyl methacrylate-co- acrylic acid-co- dimethacrylated adipic acid anhydride (25:65:10)
A mixture of 25mol% methyl methacrylate, 65mol% acrylic acid and 10mol% adipic acid anhydride was prepared. Benzoyl peroxide (0.5% w/w) was added. The resultant mixture was polymerised at 55 0 C for 1 hour in an inert atmosphere. The temperature was then ramped from 55 0 C to 75 0 C over 1 hour. The polymer was post- cured at 110 0 C for 15 minutes in an inert atmosphere, to produce a clear smooth and
biodegradable polymer.
Coagulation and histological analysis
Fresh human blood was drawn by venepuncture and aliquots of lcm 3 were placed in contact with chambers made from the range of polymers described above, prepared by the method described in examples 1 and 3. The chambers were incubated at 37 0 C for 30 minutes to ensure completion of coagulation. The clots were infused with Zambonie's fixative, impregnated with polyethylene glycol of increasing molecular weights from 400gmol "1 to HOOgmol "1 . Clots were embedded in
polyethylene glycol and 20μm histological sections were taken. Sections were stained
with DAPI and examined under a microscope. Leukocyte counts were performed in
over 30 individual microscopic fields containing the blood biomaterial interface.
Mean leukocyte populations for MMA35:AA65, MMA45:AA55m MMA55:AA45
and glass were 31±15, 31±8, 47±15, 75±18 respectively. This was a statistically significant difference between MMA35 :AA65 orMMA45:AA55 andMMA55:AA45
or glass.
Coagulation and Complement Analysis
Fresh human blood was drawn by venepuncture, anticoagulated using heparin (2 IU cm "3 ) and aliquots of lcm 3 were placed in contact with chambers made from the range of polymers described above, prepared by the method described above. The
chambers were incubated at 37 0 C for 120 minutes to ensure adequate amplification of the complement reaction. Blood specimens were transferred to centrifuge tubes containing EDTA to prevent further complement response. The specimens were centrifuged at 4 0 C and lOOOg for 15 min and the plasma supernatant collected. The plasma was analysed for C5a using a commercial ELSIA kit.
RESULTS and DISCUSSION
In order to exemplify the invention, a biocompatible polymeric material
which displays preferential platelet aggregation and initiation of coagulation whilst minimising a host complement mediated foreign body response, has been produced
and characterised.
Figure 1 and Figure 2 show the X-ray photoelectron spectra (XPS) of a series of methyl methacrylate-co-acrylic acid copolymers (MMA:AA) that have been prepared by the present inventors. These spectra show an increase in the Carbon Is binding energy shifted to 286eV represented carboxylate relative to the methyl at
283 eV indicating an increase in relative surface acid concentration along with an
increase in bulk acid concentration. An attachment study using whole blood was
performed. Blood was placed in contact with polymers containing 45%, 55%, 65%
carboxylic acids and glass, the surfaces were then fixed Zambonie's fixative.
Scanning electron microscopy of these surfaces was performed. Figure 3 shows a
section through a clot formed on a surface with 45% carboxylic acid 55% methyl methacrylate and Figure 4 shows a top down view of a clot formed on a glass surface. A comparison of these images shows that the synthetic surface directly effects the
formation of a fibrin rich coagulation plug. Figure 5 shows a histological section through the interface between a 65% carboxylic acid 35% methyl methacrylate blood
and figure 6 shows a similar histological section through a glass blood interface. Both sections are stained with DAPI which specifically stain nucleated cells. In blood the nucleated cells represent leuckocytes. Figure 7 shows the combined data of leukocyte population density at the blood material interface for MMA35-.AA65 MMA45:AA55 MMA55 : AA45 and glass. This shows a statistically significant decrease in leuckcyte concentration at the interface for the MMA35:AA65 and MMA45:AA55 when
compared with MMA55:AA45 and glass. This is indicative of a significant decrease
in the severity of the foreign body response.
Figure 8 shows plasma concentration of C5a collected from whole
heparinised blood incubated with methyl methacrylate: acrylic acid (MMA), butyl methacrylate : acrylic acid (BMA), ethyl methacrylate : acrylic acid, (ET) isobutyl methacrylate : acrylic acid (ISO) with acrylic acid concentrations of 45, 55 and 65% (for example methyl methacrylate: acrylic acid in the ratio 45:55 is MMA55) and
glass. This shows the significant effect on the initiation of complement mediated foreign body response by the combination the acid concentration and the choice co-
monomer.
Figure 9 is comparative, reproducible histological section of a blood clots formed on
a MMA35%:65%AA surface, stained with DAPI for nuclei / leukocytes from donors
b-19 and l-3.
Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope
of the present invention.
All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.
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