Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
  • A61L24/10—Polypeptides; Proteins
  • A61L24/106—Fibrin; Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/36—Blood coagulation or fibrinolysis factors
  • A61K38/363—Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/04—Macromolecular materials
  • A61L31/043—Proteins; Polypeptides; Degradation products thereof
  • A61L31/046—Fibrin; Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to the prevention of post surgical adhesions and, more particularly, concerns enhanced methods and compositions using a fibrin sealant to prevent such adhesions .
• tissue adhesions between adjacent tissues is an adverse side effect of many surgical procedures. It is believed that following abdominal surgery the incidence of peritoneal adhesion formation may be as high as 90%. These post surgical adhesions often lead to pain, discomfort, immobility and, in the case of gynecological surgery, female infertility. Also, post surgical adhesions may even result in life-threatening bowel strangulation is some instances. Thus, there exists a large unmet need for a way to prevent post surgical adhesions, given the potential discomfort and medical risks they pose following routine surgery and the accompanying financial burden of remedying this adverse effect.
• a number of pathways have been investigated to reduce or prevent post surgical adhesions.
• One proposed mechanism involves the reduction of the inflammatory reaction at the wounded site following surgery.
• corticosteroids NSAIDS, histamine antagonists and calcium channel blockers has bee suggested.
• Another prevention method recommends the inhibition of coagulation at the wounded site using, e.g., heparin or oral anticoagulants.
• Still further methods entail the promotion of fibrinolysis through appropriate administration of, for example, fibrinolysin, Streptokinase, Urokinase and t-PA.
• barrier materials have been suggested for the prevention of post surgical adhesions.
• amniotic membrane rubber, silver foil, Teflon, dextran, hyaluronic acid, Surgigel ® (regenerated cellulose) , Interceed® (TC7 oxidized, regenerated cellulose) , Polaxamer 407 (temperature dependent polymer) , Gore-Tex® (expanded polytetrafluorethylene) and SepraFilm® (hyaluronic acid derivative film) .
• fibrin sealant was found to have no statistically significant effect in preventing perivascular adhesions following arteriotemics of the femoral and carotid arteries of dogs (Dickinson, C. et al .
• fibrin sealant did not prevent adhesion formation in colonic anastomoses in the rat (van der Ham, A., et al.; J Surgical Research, 55; 256-260, 1993) and fibrin sealant did not reduce post surgical adhesions following ovarian reconstruction in the rabbit (Bilgin, T., et al.; Gynecol Obstet Invest 39; 186- 187, 1995) .
• WO 92/22312 to Wadstrom discloses combinations of fibrin sealants and biocompatible polymers reportedly useful for the prevention of post surgical adhesions.
• WO 92/22312 reports that fibrin sealants alone provide a wound healing effect which results in strong scar formation and does not prevent adhesions.
• the viscosity enhancing polymers are useful to allow application of the otherwise watery sealant components to vertical surfaces and also prevents adhesions. These polymers are high molecular polyglycors or polysaccharides .
• WO 96/22115 discloses a self supporting sheet material of cross-linked fibrin having a particular range of pore sizes to prevent or reduce post surgical adhesions.
• the use of spray application to prepare these and similar sheets is reported in WO 98/02098.
• These sheets are described as generally non- adherent and non-hemostatic and are preferably used in conjunction with a known fibrin sealant which does possess adherence and hemostatic capabilities.
• These sheets are prepared using high concentrations of fibrinogen and high dose thrombin to obtain the desired structure regarding pore size and distribution (preferably ⁇ 20 ⁇ m, more preferably ⁇ 5 ⁇ m, most preferably ⁇ 1 ⁇ m) .
• the sheet is pre-formed outside of the body and needs to be applied as a solid material, i.e., not applied as a spray or liquid.
• fibrin sealants are used increasingly for hemostasis and fluid and air leakage in surgical procedures, it is important that a sealant is used which does not promote adhesion formation. Also, since fibrin is a part of the natural healing process it seems desirable to attempt to use fibrin in resolving the post surgical adhesions problem, as well. It would be preferable to be able to accomplish this without polymer additives, without high concentrations of fibrinogen, without subjecting the patient to high dose thrombin and, preferably, without the need for preformed sheets which must, in turn, be used with standard sealants.
• a co-pending U.S. application, 60/136,901, filed concurrently herewith and entitled "Prevention of Post Surgical Adhesions Using a Fibrin Monomer Sealant” discloses that fibrin polymers, which may also be useful as fibrin sealants, are extremely useful in the prevention of post surgical adhesions when the polymer is formed of more chemically-natural materials. This is understood to refer to materials which most closely resemble the body's own clotting materials preferably patient-derived, i.e., autologous . That co-pending application describes such materials as involving one or more of the following:
• Fibrin sealants are believed to play a positive role in wound healing but prior art sealants include several factors which may overly accelerate the inflammatory response and cause post surgical adhesion formation.
• Fig. 1 Illustrates a rabbit paw flexor tendon per the experiments of Example 2.
• Fig. 2 Is a graph showing variation of dG' /dt with time per Example 2.
• Fig. 3 Is a graph illustrating the adhesion experiment of Example 3.
• Fig. 4 Is a graph comparison of VivostatTM, Tissucol® and Beriplast® kinetic rate constants based on a second order reaction over the initial 200 seconds of polymerisation.
• Fig. 5 Is a graph illustrating adhesion strength at first breaking point at various polymerisation times for of VivostatTM, Tissucol® and Beriplast®.
• Fig. 6 Is a graph illustrating elongation at first breaking point at various polymerisation times for VivostatTM, Tissucol® and Beriplast®.
• novel fibrin sealants which provoke less of any inflammatory response than prior art sealants, and methods of using such sealants, are disclosed. It has now been found that fibrin sealants can be prepared in a number of ways to selectively include or exclude components which are presently understood to upregulate or downregulate the body's natural inflammatory response.
• the novel sealants of this invention are useful in any instance where fibrin sealants have known utility. Nonlimiting examples include use in hemostasis, sealing of fluid or air leaks, delivery of cells, tissue repair and would healing, drug delivery and particularly useful in the prevention of post surgical adhesion formation.
• the present invention discloses fibrin sealants designed to produce less of an inflammatory response than their prior art counterparts. Accordingly, they reduce the incidence of post surgical adhesion formation while providing excellent hemostasis, adherence and fluid sealing benefits.
• One key to the inflammatory response is understood to be the production of nitric oxide.
• the fibrin sealants of the present invention downregulate nitric oxide formation and the inflammatory response, generally, in several ways. Each of these factors are believed to provide reduction in post surgical adhesion formation and the present invention involves methods using one, some or all of these factors.
• the fibrin polymer resulting from the present methods is able to reduce or prevent post surgical adhesions over a variety of fibrin concentrations and need not be a high concentration material when compared to the prior art. Further, there is no need to pre-form sheet-like materials ex vivo for application to a surgical wound site and no need for additional sealant layers to be used.
• the present fibrin sealant when applied using the methods described herein, possesses not only good barrier qualities to resist post surgical adhesion formation but also does less to induce their formation. At this same time it provides hemostasis, fluid sealing, adherence to the tissue and enhanced cell migration at the wound site but not into the sealant. This enhanced cell migration is believed to provide improved angiogenesis and tissue repair.
• fibrin II polymers formed by the cleavage of fibrinopeptides A and B from fibrinogen.
• U.S. 5,750,657 describes novel fibrin monomer-based sealants which can be either fibrin I or fibrin II. It has now been found that fibrin I polymer sealants provide unexpectedly improved reduction of post surgical adhesion formation when compared to prior art sealants. Fibrin I, which is the result of the cleavage of fibrinopeptide A from fibrinogen, provides less of an inflammatory response than does fibrin II. It is believed that the fibrin I downregulates nitric oxide formation when compared fibrin II polymers.
• Nitric oxide induces, and at high levels can overly accelerate, the natural inflammatory and cell migratory responses, as well as the accretion of collagen. These are essential steps to formation of post surgical adhesions. It may explain why the preferred embodiments in WO 98/02098 which utilizes a fibrin II polymer sheet (as evidenced from the combination of fibrinogen and thrombin) must be preformed and cured ex vivo to avoid formation of post surgical adhesions. With the present sealants and methods, the fibrin is applied or sprayed onto the desired site and the resulting fibrin I polymer provides hemostasis and prevents post surgical adhesions.
• the fibrin I polymer can also slowly convert to fibrin II over time, e.g., over a period of a few hours.
• Animal studies have now found that post surgical adhesions are present in at least a weak form within 10 minutes following injury and are relatively tenacious at 1 hour post-injury. It is also believed that primary fibrinous adhesions form during the first 16 hours post-injury in what can be called a post surgical adhesion modeling stage. Further, a post surgical adhesion remodeling stage occurs from 16 hours onward in which fibrinous adhesions mature into fibrous adhesions.
• the presence of a fibrin I (or substantially fibrin I) polymer during the first 10 minutes to 2 or 3 hours post-injury provides notable improvement in post surgical adhesion prevention compared to other prevention methods.
• the preferred fibrin sealant used in the present methods is initially formed as a fibrin I polymer which gradually changes to a fibrin II polymer or a fibrin I/fibrin II mix over the 2-3 hour period post-application.
• the important feature is that during the initial period when adhesion formation and potential ingrowth into the sealant are understood to occur, the polymer is preferably a fibrin I.
• part of the present invention involves fibrin sealants with reduced amounts of fibrinopeptide A and, in particular, methods of using such sealants preferably in a method of reducing the incidence of post surgical adhesion formation.
• the sealant formulation applied to the patient should contain less than 50% of the theoretical FPA expected.
• fibronectin A major player in binding these and other factors onto the fibrin polymer is fibronectin.
• Some sealants in the prior art add fibronectin to increase these levels. The natural fibronectin levels in human plasma are between 300 and 400 ⁇ g/ml.
• fibrin sealants with reduced fibronectin levels can provide a reduction in the formation of post surgical adhesions. Further, lower levels of fibronectin appear to result in a fibrin polymer with greater resistance to cell penetration. Thus, fibrin sealants with reduced levels of fibronectin are especially useful in methods where the prevention of post surgical adhesions is desired and beneficial.
• sealants with tPa are believed to enhance a more controlled inflammatory response resulting in wound healing/tissue repair, but not in post surgical adhesion formation.
• these levels of tPa are combined with the lower levels of fibronectin, as discussed above, to avoid binding the tPa onto the polymer.
• PAI-1 levels which, of course, affect the tPa levels.
• sealants in accordance with the present invention are substantially free of PAI-1.
• the present invention provides fibrin sealants which are found to promote wound healing while preventing post surgical adhesions. Given that some upregulation of the inflammatory response is believed necessary for wound healing, but too much upregulation may result in adhesion formation, it is remarkable that a single fibrin sealant product can provide both of these levels.
• sealants of the present invention can also be applied with additives, e.g., hydrocolloids, polymers, drugs and the like.
• the fibrin sealants of the present invention may also include one or more additional autologous plasma proteins.
• Autologous plasma proteins preferably selected from those which would otherwise be present in a natural clotting process can be added to the sealant components before or during application to the surgical wound site in accordance with the present methods. Alternatively, they can be coharvested in the process of producing the one or more autologous blood components which will form the fibrin sealant.
• a process to prepare autologous fibrinogen may result in a natural cocktail including the desired fibrinogen and several other proteins and/or growth factors. Rather than purify the fibrinogen so as to remove the additional autologous materials, they can simply be co-applied as a whole with the fibrinogen.
• blood components preferably autologous
• fibrin monomer may include one or more proteins which are added or co-harvested and are selected from the group consisting of prothrombin, factor XIII (activatable) , plasminogen, fibronectin, antithrombin III and factor X and which may be autologous.
• compositions useful herein include about 10-30 mg/ml of autologous fibrin monomer, about 10-40 ⁇ g/ml of autologous prothrombin and about 100-200 ⁇ g/ml of plasminogen. They may further include 5-100 ⁇ g/ml of activable factor XIII and/or 45-150 ⁇ g/ml of fibronection and/or 2.0-7.0 ⁇ g/ml of factor X and/or 50-200 ⁇ g/ml of antithrombin III, wherein all such components may be autologous to the patient receiving the anti-adhesion treatment .
• the present sealants are extremely useful for hemostasis, sealing of other fluid or air leaks, wound healing and prevention of post surgical adhesions.
• a fibrin sealant in accordance with the present methods was prepared as described by Edwardson et al. in U.S. 5,750,657 and using a process and apparatus as disclosed by Holm, inter alia , in U.S. 5,741,428, U.S. 5,603,845, U.S. 5,824,230 and U.S. 5,958,253.
• Freshly drawn anti coagulated whole blood 120ml plus 17ml 4% trisodium citrate USP was centrifugally separated and the resulting plasma (60ml) reacted with biotin-batroxobin for 10 minute at 37 °C.
• the acid soluble fibrin I polymer produced was isolated by centrifugation and dissolved in 3.5 - 5.2ml 0.2M sodium acetate buffer (pH4) containing calcium ions.
• the resulting FI monomer solution was co-applied with a carbonate/bicarbonate buffer (pHIO) in a ratio of 7:1 (Fl.pHlO) .
• Murex Lop rabbits were used for this study. They were of equal sex distribution and the body weight ranged from 2500 to 4500 grams. There was one death prior to end point assessment making the total number of rabbits 19 (9 male, 10 female) . The animals were obtained at least seven days prior to surgery from Murex BioTech Ltd (Dartfort, Kent) to allow for acclimatization. Throughout the study period they were housed in single cages and fed and watered ad libi tum . Regular assessment of the animals' general condition and surgical wound were carried out in accordance with the U.K. Home Office "Guide for the Care and the Use of Laboratory Animals" 1996.
• the left front paw' s flexor aspect was shaved with hair clippers.
• the operative site was prepared with chlorhexidine in alcohol and iodine in alcohol scrubs. The field was isolated with sterile drapes.
• the second and fourth digits of the left front paw were longitudinally incised over the base of the proximal phalanx. Blunt dissection in the midline revealed the digital sheath and its tendinous contents.
• the sheath was then opened between pulleys A2 and A3 (a point corresponding to the middle of the proximal phalanx) . Flexor digitorum profundus was exposed ( Figure 1).
• Adhesion development was assessed by the use of a tensiometer in all operated groups.
• the animals unoperated right front paw were assessed in the same way so as to provide an unoperated control group for comparative analysis. Double blind biomechanical assessment was therefore conducted on 5 groups.
• Group 5 Mobilised (digit 4) operated and no further treatment .
• the tensiometer (NE Holm A/S, Denmark) measured the force in grams required to pull the flexor digitorum profundus tendon from its sheath.
• the freshly culled animals' front left and right second and fourth digit were each dissected and the flexor digitorum superficialis and flexor digitorum profundus were transacted proximal and distal to the operative injury site.
• the proximal dissection culminated in transacting the two tendons approximately 15 mm proximal to the mouth of the digital sheath.
• the raw tensiometer pulls mean value for each group is graphically represented in graph 1. Interpretation of the raw data revealed an overall reduction in pull required to remove the tendon from its sheath by 75.6% when comparing the total treated FI monomer sealant groups. When separated into mobile and immobile the reduction from untreated to treated groups was 79.7% and 76.8% respectively.
• the boxes correspond to the interquartile range (the central 50% of the data) .
• the solid internal line represents the median value.
• the dotted horizontal line is the mean value.
• the length of the whiskers are plotted individually. Points outside these are individually graphed.
• Table 1 The geometric mean tension and 95% confidence intervals for the mean per group
• the clinical performance of fibrin sealants is influenced by physical properties such as elasticity, tensile strength, and ability to adhere to human tissue. These properties are related to the internal structure of the fibrin sealant that builds as it polymerises. Analysis of the minimum polymerisation time to achieve a functional fibrin clot is clinically important. Instant tissue-fibrin sealant adhesion is desirable to ensure that the fibrin sealant functions on contact and remains at the site of application without being washed away by blood or displaced by movement of the target tissue (e.g., the heart or lungs).
• the physical characteristics of fibrin sealants are related to the extent of fibrin crosslinking. Determination of the polymerisation rate allows calculation of the minimum time required to produce a functional clot.
• Torsion rheometry experiments were performed using a controlled stress rheometer, Carri-Med CSL 100 auto gap. Approximately 0.5 ml of fibrin sealant was applied on to the bottom of the rheometer. The top was a 2 cm diameter fine- hatched plate and the sample platform consisted of a 2 cm diameter fine-hatched plate over a Peltier baseplate. The temperature of the rheometer was set at 37 °C. Oscillation experiments were performed using a constant'' oscillation' torque of 15 ⁇ Nm at a frequency of 0.1 Hz for 11 minutes.
• Adhesion experiments were performed using a recently described model involving use of vital human tissue (Kjaergaardetal, Eur. Surg. Res. 1999).
• the human tissue samples were greater saphenous vein grafts left over from coronary artery bypass grafting. To ensure tissue vitality, all samples were kept in physiological saline solution and used within 24 hours of harvesting.
• the vein graft was split longitudinally and the split graft was cut into 1 cm 2 samples that were fixed to the sample holder using Gore-Tex V5 retaining sutures. The two samples were brought into close proximity at an angle of 45°, and 0.1 ml of fibrin sealant was sprayed on the tissue surfaces, where the adventitia was exposed.
• Adhesion strength defined as force divided by the cross-sectional area of the sample, adhesion energy (i.e., area under the experimental curve as seen in Figure 3), and elongation (i.e., extension reached by the specimen) were calculated for each experiment. Mean values were calculated at each polymerisation time.
• a single curve showing dG' /dt versus time was prepared averaging the values for the sixteen samples at each time point.
• Adhesion experiments were performed on samples of sprayed fibrin sealant in order to compare similar application systems. Most of the samples showed a first breaking point (i.e., intersection of points A and B, see Figure 3) before the maximum force was reached. This point was detected by a sudden reduction in adhesion force ( Figure 3) and was visually observed as a partial breaking of the sample. This represents the limit of the elastic character of the sealant and the first failure of the system, which has clear implications from a clinical point of view. Elongation at first breaking point (i.e., elongation at B) and adhesion strength at first breaking point (i.e., force at point A divided by the cross- sectional area) were measured for each sample.
• Adhesion strength 49.95+4.55 93.61+8.52 131.64+11.98 168.35+15.32 (gem -2 )
• Example 4 The purpose of this Example 4 is to evaluate the ability of three fibrin sealants in the prevention of post surgical adhesion (PSA) in the peritoneal cavity of rabbits following surgical injury to the uterine horn and the opposing ipsilateral peritoneal wall.
• PSA post surgical adhesion
• This example evaluate syringe- applied sealants as follows: a) the VivostatTM fibrin I monomer-based fibrin sealant as used in the earlier examples; b) Tissucol®, a fibrin sealant which was commercially available in Europe through Immuno AG in Austria, and which is a two component sealant system involving the coapplication of pooled human fibrinogen (in an aprotinim-containing solution) and a bovine thrombin component in a calcium chloride solution; and c) Cyroprecipitate (CYRO) , a cryoprecipitate concentrated blood component containing fibrinogen from a single donor and co-applied with bovine thrombin .
• CYRO Cyroprecipitate
• the Tissucol® and CRYO were all evaluated with 4 units and 500 units of bovine thrombin.
• the injured areas were then held apposed using positional sutures, placed outside the experimental site, with treated sites separated by a layer of fibrin sealant. Experimental sites were then left in vivo for the length of recovery time dictated by the study protocol. Due to the level of injury induced and injured surfaces being apposed this rabbit uterine horn abrasion model is a severe experimental PSA model, with a "worse case" scenario created.
• templates 25 mm x 3 mm, designed to fit the uterine horn or peritoneal wall, were specially constructed. Abrasions were induced using a scraper with a standard depth (1.5mm) which fitted the exact area of the template. With the additional parameter of using a set number of scrapes for each injury site, injuries were maximally standardised within the limits of biological variation.
• Uterine horns were chosen as the experimental site, together with the peritoneal wall, due to their size, structure and location within the peritoneal cavity. Heavy handling of tissues has been shown to induce PSA formation (Boys, 1942; Connolly & Smith, 1960) and hence excessive tissue handling needed to be avoided.
• ex-breeding rabbits were also chosen for these investigations due to their large size facilitating appropriate procedures.
• Premedication was administrated with hypnorm (Fentanyl citrate 0.315mg/ml and fluanisone lOmg/ml. Supplied by Janssen Saunderton, High Wycombe, Buckinghamshire.) (0.2ml/kg body weight intramuscular) to the right gluteous maximus muscle.
• hypnorm Fetanyl citrate 0.315mg/ml and fluanisone lOmg/ml. Supplied by Janssen Saunderton, High Wycombe, Buckinghamshire.) (0.2ml/kg body weight intramuscular) to the right gluteous maximus muscle.
• Surgical anaesthesia was induced with hypnorm (0.3ml/kg body weight intramuscular) and diazepam (5mg/ml diazepam. Supplied by Phoenix Pharmaceuticals Limited, Gloucester.) (2.5mg/kg body weight intravenous) .
• hypnorm 0.3ml/kg body weight intramuscular
• diazepam 5mg/ml diazepam. Supplied by Phoenix Pharmaceuticals Limited, Gloucester.
• 2.5mg/kg body weight intravenous 2.5mg/kg body weight intravenous
• Laparotomy wound closure was in two layers: first with 2/0 plain catgut on atraumatic half round needle for the peritoneum and muscle layers, using transplant longflow, overunder, non-interrupted crossed sutures; and secondly with 2/0 Prolene or similar on half round cutting needle using interrupted mattress sutures for the skin. Immediately after closure of the wound, wound dressing was applied on and around the wound.
• Mean volumes cf PSAs were 73.93mm for the control group, 1130mm 3 for the VivostatTM sealant treated group, 21.06 and 179.98mm 3 for the CRYO treated groups with 4 and 500 units thrombin respectively and 106.12 and 69.42mm 3 for the Tissucol® treated groups with 4 and 500 units thrombin respectively (Graph 3) .
• the percentage volume of PSAs for each treatment was 15.28% VivostatTM sealant, 28.49% CRYO (4 units thrombin), 243.46% CRYO (500 units thrombin), 143.55% Tissucol® (4 units thrombin) and 93.90% Tissucol® (500 units thrombin) (Graph 4).
• Vivostat fibrin I monomer based sealant demonstrated a superior prevention of PSAs in this model by providing a nearly 85% reduction in PSAs over the control and a significant improvement over the other sealants tested.
• the aim of this example was to assess the ability of VivostatTM Fibrin Monomer-Based Fibrin Sealant made from human blood (prepared as in Example 1) to reduce or prevent post surgical adhesions in a rat caecal abrasion model.
• the fibrin I solution concentrations used on the treated wounds ranged from 13.46 to 16.02 mg/ml.
• a mean volume of 0.79ml VivostatTM Fibrin Sealant was applied via the spray application to experimental sites.
• VivostatTM Fibrin Sealant is an effective agent for the reduction of post-surgical adhesions when compared to controls in this rat caecal abrasion model.
• This example is designed to evaluate the formation/prevention of PSAs m the stomach, colon and caecum of the pig by introducing a surgical injury to those sites similar to the rabbit uterine from model above.
• Pig models previously used to investigate PSAs demonstrate that pigs from PSAs in response to injury or trauma by identical pathogenesis to humans.
• control (untreated/injured) animals were compared to those treated with of VivostatTM Fibrin Monomer-Based Fibrin Sealant as prepared in Example 1, but wherein the sources were (a) human and (b) from the pig being treated, i.e., autologous sealant.
• Keta ⁇ une Kerar- 50mg/ml ketamine hydrochloride. Supplied by Parke-Davis, Pontypool, Gwent . ) (5mgs/Kg) plus Xylazme (Rompun 2%-Xylaz ⁇ ne hydrochloride 23.32mg/rnl (equivalent to 20mg/ml xylazme) and IMg/ml methyl 4-hydroxy-benzoate (preservative) . Supplied by Bayer Pic, Animal Health Business Group, Bury St. Edmunds, Suffolk.) (lmg/kg)) intramuscularly in the gluteous maximus muscle. At the operating suite, pigs were induced to and maintained at full anaesthesia with Halothane at 4% in oxygen and nitrous oxide delivered at 1.5 litres per minute and 0.5 litres respectively per minute via mask to the snout.
• anaesthetised pigs were transferred to lay in the supine position on the operating table, where they were secured by soft tapes to each limb.
• a plastic ear tag bearing the unique pig identification number for the study was secured to one of the animals' ears.
• the surgical site was shaved and scrubbed with chlorhexidine in alcohol followed by swabbing twice with iodine in alcohol. Sterile operative procedures were adhered to from this point on.
• the animals were draped. Cutting diathermy and scissors were used to expose the spiral colon and the stomach through a midline laparotomy starting at the level of the distal xyphoid process of the sternum and extending distally 10 - 12cms.
• sutures passed only through the internal serosa of the peritoneum and then passed through the serosa at the respective en ⁇ s of the apposed abraded areas on the stomach or colon. Sutures were tightened to bring the two apposed abraded areas close together but not in contact.
• human or porcine fibrin sealant was applied to the abraded areas (mean volume of 1.3ml per experimental site), whereas no treatment was applied in control cases. Positional sutures were then tightened to bring the two abraded areas into close contact.
• the laparotomy was closed in two layers, first with 2/0 Dexon Plus on an atraumatic half round needle for peritoneum and muscle layers, second with 2/0 Prolene or similar on half round cutting needle for skin.
• the first suture layer was of "transplant" longflow, over-under, non-interrupted crossed sutures, the second layer was of interrupted cruciate mattress sutures .
• Resected tissues were trimmed of excess adipose tissue, pinned flat on stiff card, to retain a standard, lifelike tissue positional relationship, and immersion fixed in 10% neutral buffered formal saline for at least 24 hours at room temperature .
• Mean volume of PSAs for the control group was 207.25mm 3 for the colon, 31.33mm 3 for the caecum and 248.11 mm 3 for the stomach with an overall mean of 181.78mm 3 .
• Human fibrin sealant treated group demonstrated mean volumes of 42.83mm 3 , 8.96mm 3 and 204.47mm 3 for the colon, caecum and stomach, respectively, with an overall volume of 74.77mm 3 .
• Figure 8.5 experimental sites treated with porcine fibrin sealant, with 6.25mm 3 for the stomach and a group mean of 1.27mm 3 (Graph 5).

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