WO2017123109A1

WO2017123109A1 - Tissue adhesive composition, a method of producing it, and its application

Info

Publication number: WO2017123109A1
Application number: PCT/PL2017/050002
Authority: WO
Inventors: Renata WAWRZASZEK
Original assignee: Uniwersytet Medyczny Im. Piastów Śląskich We Wrocławiu
Priority date: 2016-01-12
Filing date: 2017-01-10
Publication date: 2017-07-20
Also published as: PL229593B1; PL415767A1

Abstract

The subject of the present invention is a new tissue adhesive composition, a method of producing it and its use in daily surgical practice, particularly for occluding and sealing insufficient veins or congenital vascular anomalies.

Description

Tissue adhesive composition, a method of producing it, and its application

The subject of the present invention is a new tissue adhesive composition, a method of manufacturing and its application in daily surgical practice, particularly for the occlusion of insufficient venous vessels or congenital malformations.

Chronic venous insufficiency is presently a social problem in developed countries. It is estimated that in Great Britain alone the cost of venous ulcers is about 400-600 million pounds, and over a billion in the USA. Studies conducted in Western Europe show that lower limb varicose veins afflict 35-53% of the population, 25-33% of women and 10-20% of men. In Poland, in 2003, a multi-site study on chronic venous insufficiency of all stages of advancement related to almost half of all Poles, women more frequently: 50.99%. Lower limb varicose veins were noted in 34% of subjects, and in 25% of cases in both limbs.

Chronic venous insufficiency is the disruption of superficial or deep veins. It is caused by the insufficiency of venous valves. It may be a congenital or acquired illness. The insufficiency can concern superficial veins, the side branching's conjoining them, perforators or deep veins. Symptoms which announce venous insufficiency are calf cramps, numbness, tingling, burning sensation, oedema, leg pain, heavy legs, nocturnal leg cramps, and restless legs syndrome. Venous illness is a cause of workplace absenteeism, long term workplace disability, it can contribute to early disability pension and may also lead to thrombosis and pulmonary embolism, which carry a risk of acute death.

Tissue glues used in medicine are seen as the most advanced method of attachment. Tissue glues can constitute an effective alternative in clinical practice in cases where other methods are not applicable due to contraindications. The use of tissue adhesive eases procedure and shortens hospitalization time. Despite the need in various areas of medicine, the choice of tissue adhesives is still small. Moreover, adhesives, despite many promising clinical results, are not without problems. For example, they induce inflammation and degrade in the moist biological environment.

US7449498 reveals adhesive composition for veins encompassing n-butyl-2-cyanoacrylate and beta-tricalcium phosphate mixed in a 1 :4 or 1 :7 ratios.

US3667472 discloses the surgical use of monomeric C2-C4 alpha-cyanoacrylate adhesive. The problem of this class of adhesive is their unsuitability for use in internal organs, and thereby in anastomoses, due to its well documented toxic and oncogenic properties.

Prior art (US6174919) discloses an n-butyl-2-cyanoaciylic tissue adhesive (Histoacryl®). Despite many studies on the use of cyan acrylic adhesives there are many controversial findings related to its use, safety and efficacy of treatment. The n-butyl-2-cyanoacrylate glue polymerises in contact with living tissue, and polymerizes rapidly with an anionic mechanism. Its polymerization time is dependent on the type of tissue (does not flow in the presence of blood and bodily fluids), and reaches maximum mechanical durability in 60-90 seconds. During polymerization, the temperature is no higher than 45°C (considerable local temperature increases at the sites of adhesion take place). The post-curing joint is hard, inelastic and adhesives of this kind quickly initiate haemostasis. They are highly brittle and the adhesive layer is eliminated through hydrolysis. It is used for very small gaps, max. 0.15 mm. The thicker the adhesive layer the less complete is its polymerization. The n-butyl-2- cyanoacrylate glue is incapable of filling any gaps and may also be difficult to apply to wounds of a large surface area. During application, n-butyl-2-cyanoacrylate adhesive sticks to gloves and surgical tools which impedes its application and often hardens while it's still being dosed. An excessive quantity of adhesive and its large dilution with lipiodol may promote circulatory thrombosis, whereas its excessively rapid delivery can promote thrombotic complications. Moreover, n-butyl-2-cyanoacrylate adhesive is characterized by a sharp, acrid odour which is highly perceptible in dry air. It acts as a respiratory irritant, and the fumes may induce drowsiness and dizziness.

Literature contains examples of complications due to the use of n-butyl-2-cyanoacrylate, and thrombosis complications, often very serious, have been observed. Individual cases of pulmonary, spleen, portal vein embolisms, thromboembolisms in the portal and splenic veins, brain thromboembolisms, as well as in the coronary vessels and spleen were observed. One case is known of a thromboembolism in the left kidney artery, inferior vena cava, and in parallel, in the pulmonary artery. The patient also exhibited a septicaemia complication in the form of pneumonia due to pulmonary infarction and recurrent severe kidney ulcers. Septicaemia and complications were observed in patients with pulmonary embolisms. Very difficult, terminal cases occur sporadically.

To summarise, there is a deeply felt need to formulate an improved composition of tissue adhesive or sealant that eliminates the prior art problems and may be used in daily surgical practice to occlude and seal insufficient vessels of the venous system.

The goal of the present invention is to design an adhesive formulation that would minimize the deleterious effects of currently used adhesive substances. The goal of the present invention is to deliver a composition of medical adhesive in which the components are selected quantitatively and qualitatively such that the adhesive joint facilitates the elastic and permanent adhesion to the vein walls. The technical problem solved by the present invention is to deliver a composition of medical adhesive for the occlusion and sealing of insufficient vessels of the venous system or congenital vascular anomalies. The composition will allow for the anchoring of the adhesive substance in the pores and irregularities of the joined surfaces, and for its cohesion, such that a strong joint is formed characterised by impermeability, good physio-chemical properties (durability, hardness, elasticity) and resistance to enzymes.

The subject of the present invention is a tissue adhesive composition for occluding or sealing insufficient vessels of the venous system or congenital vascular anomalies comprising:

a) from 85 - 86% by weight poly(methyl-vinyl-siloxane),

b) 5.4 - 6.5% by weight silicon dioxide,

c) 8.5 - 8.6%) by weight crosslinked,

wherein the ratio of poly(methyl-vinyl-siloxane) to silicon dioxide is from 13 : 1 to 16: 1.

Preferably, the viscosity of poly(methyl-vinyl-siloxane) is 18 000 mPa-s - 23 000 mPa-s at a temperature of 25°C.

Preferably, the BET surface area for the silicon dioxide is 175-225 m²/g.

Preferably, the cross linker is a platinum catalyst.

Preferably, the medical adhesive composition contains 85%> by weight poly(methyl-vinyl- siloxane), 6.5%> by weight silicon dioxide, 8.5%> by weight platinum catalyst.

The subject the present invention is also a method of producing a composition of tissue adhesive for occluding or sealing insufficient vessels of the venous system or congenital vascular anomalies as above, comprising:

a) mixing poly(methyl-vinyl-siloxane) with silicon dioxide at a ratio of 13 : 1-16: 1, b) homogenizing the mixture,

c) setting aside the mixture for 12 h,

d) adding cross linker,

e) homogenizing the mixture until obtaining an adhesive of homogenous consistency. Preferably, before being added to the binder the silicon dioxide is dried for 48h at a temperature of 50°C in a laboratory drier.

Preferably, the cross linker is a platinum catalyst.

The subject the present invention is also a tissue adhesive composition defined above for use in treatment of insufficient vessels of the venous system or congenital vascular anomalies. An adhesive a composition according to the present invention is denoted as R 88 herein. The base of the adhesive is a poly(methyl-vinyl-siloxane) binder, encompassing in its general structure functional groups directly bound to the siloxane: methylene groups, hydroxyethyl groups (viscosity of 18 000 mPa - 23 000 mPa at a temperature of 25°C), moreover the new composition contains silicon dioxide (the BET surface area 175-225 m²/g) and a cross linker - a platinum catalyst.

A glue according to the present invention is a synthetic adhesive, and therefore excludes the risk entailed by the presence of human or animal components (bacteria, viruses, allergies). The resulting adhesive cures by reacting with moisture (first, a "crust" is formed on the surface, and then the curing process progresses into the interior of the joint, and the reaction is additionally accelerated by the increased temperature of 37°C, whereas the time-to-cure is slightly elongated by a lower moisture content. The adhesive composition is capable of linking at a temperature of 37°C, and has excellent thermal stability when hardened. A glue according to the present invention is highly elastic and has compensatory properties, which means that the site of adhesion is not damaged even during compression loads and returns to its position when the load is removed. The resulting product is clear, odourless, and does not exhibit either delamination or moulding. When applied gradually, adhesive R 88 is capable of entirely filling the vein lumen, adapting to the shape, and penetrates porosity due to its capillary nature. When introduced into the vein, it initiates a complex reaction between blood components and the adhesive' s surface, undergoing polymerization in the moist environment. It is retained in the vein and is not ejected out of it by the blood, at the same time staunching haemorrhaging from the vein. The cured adhesive joint effectively forms a seal, while maintaining its elasticity.

The present solution delivers an adhesive joint encompassing material factors (structure and adhesive properties), technological (a method of preparing and applying the adhesive mass), curing conditions for the adhesive joint, structural (loading, joint shape), working conditions for the adhesive (aggressiveness, temperature range, magnitude and character of stresses). Additionally, the formulated and produced glue for binding surfaces exhibits elasticity such that the glued structures can deform in accordance with their natural behaviour in the human or animal body.

The medical glue according to the present invention fulfils the criteria required from materials for implantation, and at the same time is characterised by the durability of the adhesive- surface interface, and exhibits good adherence while maintaining appropriate elasticity and durability in a biological environment.

The formulated adhesive can be used for occluding and sealing insufficient vessels of the venous system. Description of figures

Fig. 1 Sealing veins. The procedure of inserting the adhesive into a vessel is performed gradually using a calibrated gun-like dosing applicator. The glue is applied into the lumen of the varicosity using a special catheter set. This facilitates the controlled occlusion of the insufficient venous vessel, (http://www.centerforvein.com/blog/new-treatment-methods-in- phlebology, nasmed.com.pl).

Fig. 2 Plot of the changes in pH of adhesive R 88 in Ringer's solution over 33 months of incubation.

Fig. 3 Comparison of MTT results shown as the average cell vitality in the adhesive vs. a control, which was set to 100%.

Fig. 4 Comparison of the relative viability of cells in contact with adhesive R 88 and the binder for a dilution series of conditioned medium (100%, 50%, 25%, 12.5%).

Fig. 5 Relative viability index for cells in a dilution series of conditioned medium (0.2%, 0.15%, 0.1%, 0.05%).

Fig. 6a and 6b In vitro results, microscopic evaluation of the adhesive binder: [7] extract with a concentration of 100%; [8] extract with a concentration of 50%; [9] extract with a concentration of 25%; [10] extract with a concentration of 12.5%, [1 1] positive SLS sample (sodium dodecyl sulphate, vel. sodium lauryl sulphate) 0.2mg/ml, [12] positive SLS sample (sodium dodecyl sulphate, vel. sodium lauryl sulphate) 0.15mg/ml, [13] extract with SLS at O. lmg/ml, [14] positive SLS sample (sodium dodecyl sulphate, vel. sodium lauryl sulphate) 0.05 mg/ml, [15] control, [16] binder edge, [17] underneath the binder, [18] underneath the binder, culture following 24h of incubation.

Fig. 7a and 7b In vitro results, microscope evaluation of adhesive R 88: [19] cytotoxicity evaluation results of adhesive R 88 extract with a concentration of 100%, [20] extract with a concentration of 50%, [21] extract with a concentration of 25%, [22] extract with a concentration of 12.5%, [23] positive SLS sample (sodium dodecyl sulphate, vel. sodium lauryl sulphate) 0.2 mg/ml, [24] positive SLS sample (sodium dodecyl sulphate, vel. sodium lauryl sulphate) 0.15 mg/ml, [25] cytotoxicity evaluation results of adhesive R 88 extract with a concentration of SLS O. lmg/ml, [26] positive SLS sample (sodium dodecyl sulphate, vel. sodium lauryl sulphate) 0.05 mg/ml, [27] control, [28] edge of the adhesive R 88, [29] underneath adhesive R 88, [30] underneath the adhesive, culture following 24h of incubation. Fig. 8 Microscopic image of a rabbit ear surface, the vein completely filled with adhesive R 88 after 90 days post-implantation. Fig. 9a Macroscopic image following the operation after 14 days post-gluing the marginal vein of the rabbit ear with adhesive R 88.

Fig. 9b Control group after 14 following the operation - cyan acrylic tissue adhesive (Histoacryl®), Macroscopic image after 14 days following the operation of gluing the gluing the marginal vein in a rabbit ear.

Fig. 10a Macroscopic image after 90 days following gluing the marginal vein in a rabbit ear with adhesive R 88.

Fig. 10b Control group: Macroscopic image after 90 days following the operation of gluing the marginal vein in a rabbit ear with Histoacryl® tissue adhesive.

Fig. 11 - 13 Histological evaluation of the adhesive.

Fig. 11a Results after 2 weeks for adhesive R 88, Histoacryl® control adhesive) Microscopic image of healthy skin with surrounding tissues, cartilage and vasculature. Erythrocytes are visible in the vascular lumen. HE staining, mag. lOOx.

Fig. 1 lb Histology results following 2 weeks, image demonstrating the behaviours or the vein beside the site glued with adhesive R 88, Histoacryl® (control adhesive), Microscopic image of healthy skin with surrounding tissues, cartilage and vasculature. Erythrocytes are visible in the vascular lumen. HE staining, mag. lOOx.

Fig. 12a Histology results after 30 days for a vein with adhesive R 88 and Histoacryl® (control adhesive) Microscopic image of healthy skin with surrounding tissues, cartilage and vasculature. Erythrocytes are visible in the vascular lumen. HE staining, mag. lOOx.

Fig. 12b Comparison of the vein adjoining the site of gluing with adhesive R 88 and Histoacryl® (control adhesive) after 30 days. Microscopic image of healthy skin with surrounding tissues, cartilage and vasculature. Erythrocytes are visible in the vascular lumen. HE staining, mag. lOOx.

Fig. 13a Histology results after 90 days for a vein occluded with adhesive R 88 and Histoacryl® (control adhesive), Microscopic image of healthy skin with surrounding tissues, cartilage and vasculature. Erythrocytes are visible in the vascular lumen. HE staining, mag. lOOx.

Fig. 13b Comparison of the vein adjoining the site of gluing with adhesive R 88 and Histoacryl® (control adhesive) after 90 days. Microscopic image of healthy skin with surrounding tissues, cartilage and vasculature. Erythrocytes are visible in the vascular lumen. HE staining, mag. lOOx.

Examples Example 1. Production of the medical adhesive

The adhesive binder (viscosity of 18 000 mPa'S - 23 000 mPa-s at a temperature of 25°C) was mixed with silicon dioxide (BET surface area 175 - 225 m²/g), the entirety is homogenized over 60 min at 300 RPM with a mechanical laboratory mixer, and then set aside for 12h, where after the platinic cross linker is added. The entirety is homogenized after adding all of the mentioned components in the given amounts and sequence for a further 5 minutes at about

50 RPM until obtaining an adhesive of homogenous consistency.

This adhesive contains no softeners, plasticizers, nor the like, nor stabilizers.

Before being added to the binder, the silicon dioxide is dried in for 48h at a temperature of

50°C in a laboratory drier.

All viscosities described herein are magnitudes of dynamic viscosity at 25°C measured using a known method, as a shear gradient representative for its use.

Example 2. Evaluation of external appearance

The evaluation of the external appearance and properties was performed using visual inspection immediately following the production of the adhesive -visual evaluation (Tablel)

Table 1

Glue Colour Consistency Odour Foreign

contaminants

R_88 Clear Homogenous, non-stratifying, non- None None

clumping, no coagulated, unground

components, easily applied and spread,

no clods

Example 3. Evaluation of the stability of the adhesive mass (organoleptic properties)

The method consists of the visual evaluation of the adhesive solution (like for evaluating the solubility of the adhesive), samples of the adhesive were enclosed in sealed glass containers and stored at room temperature. After 24 hours, we evaluated the state of the adhesive mass - whether it did not stratify. The stability of the adhesive mass is shown in Table 2.

Table 2

Glue Stability of the adhesive mass Joint

R_88 No stratification, the adhesive joint is smooth, Elastic

No cracks and wrinkle Example 4. Hardness evaluation

Each sample was evaluated at 5 sites, at distance intervals of at least 5 mm. Hardness was measured after 3 s from the application of the hardness meter to the sample. The hardness result is an arithmetic mean of all of the measurements. The difference between the individual measurements cannot exceed more than ±2 Shore units. Shore type A hardness is shown in Table 3.

Table 3

Example 5. Evaluation of the stability of the adhesive mass (organoleptic properties)

The method consists of the visual evaluation of the adhesive solution (like for evaluating the solubility of the adhesive), samples of the adhesive were enclosed in sealed glass containers and stored at room temperature. After 24 hours, we evaluated the state of the adhesive mass - whether it did not stratify (Table 4).

Table 4

Example 6. Long term degradation processes

Lon term evaluation of the tissue adhesive consisted of incubating the tissue adhesive for 33 months in a warmer for 37°C in Ringer's solution (11 of solution contains: sodium chloride - 8.60 g/1, potassium chloride - 0.30 g/1, calcium chloride 0.48 g/1, ionic concentrations: Na⁺ 147.16 mmol/1, K⁺ 4.02 mmol/1, Ca⁺²2,19 mmol/1, CI^" 155.56 mmol/1) and distilled water. The ratio of solution volume to sample mass was greater than 30: 1.

Stability changes of the formulated adhesive samples were monitored by measuring pH in the Ringer's solution and distilled water. The measurements were made using an Elamtron CP- 315 pH-meter, which was calibrated before every measurement. The results show that adhesive samples in the Ringer's solution simulating a living organism at a temperature of 37°C do not alter the pH throughout the entire time of the degradation study. These results are in accordance to the ISO 10993-12 norm. The evaluated material is stable and behaves very stably in a physiological fluid environment. The samples taken therefrom were clear, colourless and odour-free.

Example 7. Cytotoxicity results

Cytotoxicity evaluation: direct test

The study made use of 12-12-good plates (Nunc), which were inoculated with 1 ml cells suspended in full culture medium (lxlO⁵ cells/ml). After 24 hours of incubation (37°C, 5% C0₂ ) the cell layer was overlaid with fragments of adhesive with a surface area no less than 10% of the good surface. After 24 h of incubation, we evaluated morphological changes in the culture using a microscope. The test was repeated 5 times and conducted in accordance with norm ISO 10993-5:2009(E).

Preparation of extracts

To prepare extracts, we used material at a rate of 3 cm² surface/lml medium with serum. The extracts were incubated for 24h at a temperature of 37°C. We prepared the following extract dilution series: 100%, 50%, 25%, and 12.5%.

Cytotoxicity evaluation: indirect test, MTT

Tissue glues that come into contact with blood must be characterized by biocompatibility, though to some degree they always interact with platelets and act on thrombosis and fibrinolytic factors. The negative effect of this activity is clotting, which may lead to the occlusion of the vein lumen or constitute a source of thromboembolic complications. The viability of cells in the conditioned medium makes it possible to analyse the interaction of the evaluated adhesive with the culture medium simulating the bodily fluids, and makes it possible to predict the behaviour of the evaluated adhesive during prolonged contact with bodily fluids and to evaluate its effect on the biological response.

The evaluation was performed on murine fibroblast line L929 (21 passage). The culture was conducted under standard conditions (37°C, 5% C0₂) in an incubator (Thermo Scientific) in MEM medium (General Chemistry Laboratory, IITD PATE, Wroclaw). The medium was supplemented with FBS (Lonza)- 10%, and L-glutamine with streptomycin (lOmg/ml) and penicillin (10,000 U/ml) (Sigma)-1%, in accordance with recommendations. The cultures were performed in bottles of 75cm² (Falcon). The cells were passaged using 0.25% trypsin in EDTA (Sigma), and PBS (Lonza). 96-well plates were used (Falcon). 96-96-good plates (Falcon) were inoculated with cells in 100 μΐ of medium at 1·10⁵ cells/ml (1·10⁴ cells/well). Each extract was repeated n = 12 times, and control n = 24. To evaluate the cytotoxicity of the extracts, we used the MTT assay (Sigma) conducted after 24 h of incubating cells with extracts. After the incubation, the medium was decanted and each well was supplemented with 50 μΐ MTT solution in MEM (lmg/ml) without supplements and with no phenyl red (General Chemistry Laboratory, IITD PATE, Wroclaw. After 2 hours of incubation, the solution was decanted and each well was loaded with 100 μΐ isopropanol. After 0.5 h of incubation, we measured absorbance at 570 nm (Epoch, Biotech spectrophotometer), and evaluated cell viability in percent (V%) in relation to the control, which constituted 100%, as per the following equation:

V = (P_b/P_k)-100% [%]

where: P_k- control sample, P_b- evaluated sample.

The test was performed in accordance with norm ISO 10993-5:2009 (E).

The MTT assay facilitates the quantitative evaluation of the cytotoxicity of the evaluated compounds. The test is based on the conversion of yellow tetrazolium salt (MTT) in the mitochondria of living cells to a purple derivative - formazan. Toxicity degrees for the test using the extracts (as per PN-EN ISO 10993-5:2009) are shown in Table 5.

Table 5

In accordance with PN-EN ISO 10993-5:2009, cell viability at 70% or less and changes in the cell culture in excess of stage 2 indicate the cytotoxicity of the biomaterial. For the binder, which is the chief component of the tissue adhesive and is conditional to its desired adhesiveness and the mechanical durability of the joint, after its curing, we observed the inhibition of cell growth to 66.70%, and did not observe cell lysis beyond stage 2.

Adhesive R 88 exhibited a cell viability of 96.92%.

An important benefit of the composition of the adhesive according to the present invention is its non-toxic character, which enables the composition to be used as an internal adhesive, without fear of safety, because literature data shows that GRF adhesive compositions have achieved limited success due to the use of hot gelatine solutions and in many cases tissues are irritated through the use of aldehydes. Intravenous fibrin glues can lead to thromboembolisms and systemic intravascular clotting. In rare cases there are allergic reactions (perceived needling and stinging at the site of use, bronchospasm, bradycardia, shortness of breath, shivers, hot flashes, headaches, rashes, hypotension, nausea, drowsiness, itchiness, anxiety, tachycardia, pins and needles, obstructed breathing, vomiting, thoracic pain, wheezing). In rare cases, these escalate to strong anaphylactic reactions, and there is also a risk of transferring viral infections. The cytotoxic activity of the adhesive binder is shown in Table 6. The cytotoxic activity of the adhesive R 88 is shown in Table 7.

Table 6

Table 7

Example 8. Animal tests

The properties of the formulated adhesive were tested on animals (permit obtained from the Local Ethics Committee in Wroclaw, decision No. 60/2012 dated October 17, 2012 for experiments on 20 albino rabbits). The tissue glue was preclinical tested in animals - albino rabbits of both sexes, weighing ca. 3200-3500 g. The animals were housed and fed in identical conditions (separate room in standard laboratory cages under controlled humidity (45-55%) and air temperature (18-25°C). They were given free-choice access to pelleted chow for rabbits and water.

Prior to the study, the R 88 adhesive was UV sterilized before the operations. The tissue glue Histoacryl - blue constituted the comparative model for the formulated adhesive R 88. Histoacryl - blue is used in the ablation of varicosities; both haemorrhaging and non- haemorrhaging ones. The rabbits were injected in the marginal vein of the ear with adhesive at a rate of 0.5 ml (0.7· 30 micro point needle).

Example 8.1. Study using scanning el (SEM)

The microstructure of the surface of the formulated adhesive and its behaviour at the site of implantation, a vein in the rabbit ear, were evaluated using a Phenom Pro TM SEM. We performed a series of images of the vein with the adhesive over 14, 30 and 90 days' post- implantation.

Microscopic observations helped examine the behaviour of the adhesive applied into animal's ear. SEM photos of the marginal vein filled with the adhesive yield an image of an adhesive joint without cracks, wrinkles, no delamination of the polymerized adhesive throughout the duration of the experiment. R 88 has good adhesion to the vein wall by forming a strong cohesive bond. As the vein is deformed and torn vein, the destruction does not take place in the adhesive layers, but in vein. At 14, 30 and 90, the adhesive was a hard polymerized mass, tightly coupled to the wall of the vein. The samples of implanted adhesive observed using SEM facilitates estimation of the erosion caused by the biological environment. There were no degradation processes in the adhesive (no micro-cracks, cracks, delamination, blisters and voids - scavenging effect of chemicals associated with the separation of low molecular weight gaseous products, no peeling adhesive). The adhesive seam did not delaminate, which could result in the reduction of mechanical strength and degradation.

Example 8.2. Post-operative results

In the postoperative period, the animals did not receive any medication. During the first days after the operation, a slight reddening can be observed in the skin surrounding the wound in both cases. 7 days' post-surgery, the vein filled with R 88 adhesive is suppler, less tense and flushed, and does not become more sensitive to the touch than immediately after surgery. The healing of wounds within the ear progressed quickly. Under the skin, it was possible to sense a thickened area where the elastic mass formed a durable seal. In no case was there any infection at the implantation site, nor oedema, signs of inflammation and abscesses at the site, nor were there inflammatory changes in the skin nor discolorations. There were no allergic or toxic reactions. Palpable hard, elastic adhesive lumps combined strongly with the vein, and there was no movement of the adhesive within the vein itself, causing asymmetry of the implant site.

After 14 days of observation, there were no local inflammatory reactions in the tissue, there was no ear oedema, scaling, nor severe irritation or necrosis. No allergic signs appeared around the surroundings of the implantation site (erythema, vesicles, papules), no itching of the areas around the implanted site, and likewise no local inflammatory reactions in tissue. There was no swelling, peeling, nor severe irritation or necrosis.

Example 8.3. Histology results

We prepared microscope slides encompassing the implanted materials with surrounding tissue using the collected fragments of ears at 14, 30 and 90 days from the injection of the adhesive. The downloaded material was subjected to a histological evaluation. This test material was fixed for 48 hours in a 5% aqueous solution of formaldehyde neutralized with a phosphate buffer at room temperature. The samples were placed in first in 70% alcohol and then 96%. Such tissue sections were freed of spurious components. The next stage was to dehydrate them three times in an acetone series: three baths for 90 minutes at a temperature of 60 °C and two more baths: carboxyl and xylene of 45 minutes each at room temperature. Following this preparation, the tissue was embedded in paraffin blocks.

A Leica 2125RT microtome was used to cut thick sections of about 4 μπι. The preparations were stained with Haematoxylin (Van Gieson (VG) method). We obtained 100 histological sections, which were then analysed microscopically using an Axioscop light microscope (Zeiss) at different magnifications, and characteristic histological images were documented photographically.

A comparison of the behaviour of the veins glued with R 88 and Histoacryl® (control adhesive used on bleeding and non-bleeding varicose veins). The results of histologic examination were identical at 14, 30 and 90 days after surgery. All histological images showed normal skin, cartilage tissue and blood vessels in the vascular lumen. There were groups of erythrocytes visible. A comparison of the behavior of living tissue adjoining the site of gluing with adhesive R 88 and Histoacryl® after 14, 30, and 90 days showed normal skin, cartilage tissue and blood vessels with a slightly wider lumen. The vein lumen showed numerous erythrocytes. The composition was found to have very useful properties in experiments conducted in animals and is well tolerated without side-effects and adverse reactions after 90 days from the operation.

Claims

1. Tissue adhesive composition for occluding or sealing insufficient vessels of the venous system or congenital vascular anomalies comprising:

a) from 85 - 86% by weight poly(methyl-vinyl-siloxane);

b) 5.4 - 6.5% by weight silicon dioxide;

c) 8.5 - 8.6%) by weight crosslinked;

2. A composition according to Claim 1, characterised in that the viscosity of the poly(methyl-vinyl-siloxane) is 18 000 mPa-s - 23 000 mPa-s at a temperature of 25°C.

3. A composition according to Claim 1, characterised in that the BET surface area for the silicon dioxide is 175-225 m²/g.

4. A composition according to Claim 1, characterised in that the crosslinked is a platinum catalyst.

5. A composition according to Claim 1, characterised in that it contains 85% by weight poly(methyl-vinyl-siloxane), 6.5% by weight silicon dioxide, and 8.5% by weight platinic catalyst.

6. A method of producing a composition of tissue adhesive for occluding or sealing insufficient vessels of the venous system or congenital vascular anomalies as defined in Claim 1, comprising:

c) setting aside the mixture for 12 h,

d) adding cross linker,

e) homogenizing the mixture until obtaining adhesive of homogenous consistency.

7. A method according to Claim 6, characterised in that before being added to the binder the silicon dioxide is dried for 48h at a temperature of 50°C in a laboratory drier.

8. A method according to Claim 6, characterised in that the crosslinked is a platinum catalyst.

9. Tissue adhesive composition as defined in Claim 1 for use in treatment of insufficiencies of veins or congenital vascular anomalies.