HEMOSTATIC COMPOSITIONSFIELD OF THE INVENTIONThe present invention relates to hemostatic compositions and processes for makingsuch compositions.
BACKGROUND OF THE INVENTIONHemostatic compositions in dry storage-stable form that comprise biocompatible,biodegradable, dry stable granular material are known e.g. from W098/008550A orW02003/007845A. These products have been successfully applied on the art forhemostasis, Floseal® is an example for a highly effective haemostatic agent consisting of agranular gelatin matrix swollen in a thrombin-containing solution to form a flowable paste.
Since such products have to be applied to humans, it is necessary to provide highestsafety standards for quality, storage-stability and sterility of the final products and thecomponents thereof. In addition, manufacturing and handling should be made as convenientand efficient as possible.
A successful product in this field (the Floseat® product mentioned above) utilizes agelatin matrix used in conjugation with a reconstituted lyophilized thrombin solution. Thegelatin matrix is applied as a flowable granular form of gelatin and thrombin with a gelatincontent of about 11 to 14.5%. Lower gelatin content results in a runny product withdiminished performance due to difficulties in having the product remain at the treatment site,especially under conditions of high blood flow. Higher gelatin particle concentration leads to aproduct that is difficult to deliver by usual means of administration, such as syringes orcatheters, due to higher resistance to flow. The inclusion of plasticizers in the composition,e.g., polyethylene glycols, sorbitol, glycerol, and the like has been suggested(EP0927053B1) and can diminish extrusion force, but inclusion of these materials does notnecessarily improve performance.
It is an object of the present invention to provide a hemostatic composition based ona crosslinked gelatin with improved adhering and hemostatic properties compared to thegelatin products such as Floseal according to the prior art and methods for making suchhemostatic compositions. The compositions should also be provided in a convenient andusable manner, namely as a flowable paste usable in endoscopic surgery and microsurgery.
The products must have an extrusion force of 40 N or below, preferably below 35 N,especially preferred below 20 N. The products should preferably be provided in productformats enabling a convenient provision of "ready-to-use" hemostatic compositions, whichcan be directly applied to an injury without any time consuming reconstitution steps.
SUMMARY OF THE INVENTIONTherefore, the present invention provides a hemostatic composition comprisingcrosslinked gelatin in particulate form suitable for use in hemostasis, wherein thecomposition is present in paste form containing 15.0 to 19.5% (w/w) crosslinked gelatin,preferably 16.0 to 19.5% (w/w), 16.5 to 19.5% (w/w), 17.0 to 18.5% (w/w) or 17.5 to 18.5%(w/w), more preferred 16.5 to 19.0% (w/w) or 16.8 to 17.8% (w/w), especially preferred 16.5to 17.5% (w/w), and wherein the composition comprises an extrusion enhancer, especiallyalbumin.
The invention also refers to the use of this hemostatic composition for treating aninjury selected from the group consisting of a wound, a hemorrhage, damaged tissue and/orbleeding tissue comprising administering such a hemostatic composition and kits makingsuch a hemostatic composition for the treatment of such injury.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTIONThe present invention provides a hemostatic composition comprising crosslinkedgelatin in particulate form suitable for use in hemostasis, wherein the composition is presentin paste form containing 15.0 to 19.5% (w/w) crosslinked gelatin (= weight of dry crosslinkedgelatin per weight of final composition), preferably 16.0 to 19.5% (w/w), 16.5 to 19.5% (w/w),17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more preferred 16.5 to 19.0% (w/w) or 16.8 to17.8% (w/w), especially preferred 16.5 to 17.5% (w/w), and wherein the compositioncomprises an extrusion enhancer.
It has been surprisingly found within the course of the present invention that theprovision of extrusion enhancers, such as albumin in the appropriate amount, enables theuse of higher gelatin concentrations and that the use of higher gelatin concentrationsimproves the hemostatic properties of such products. This is an effect which is not suggestedin the prior. Moreover, it was surprising that higher concentration of crosslinked gelatin resultin better adhesive properties (in contrast to the results known in the prior art (e.g. Fig. 4 ofW02008/076407A2).
For enabling the preferred properties due to the higher gelatin concentrations in thepaste according to the present invention, it is necessary to provide the extrusion enhancersin appropriate amounts. The amounts shall be high enough so as to obtain the extrusioneffect, i. e. to enable a flowable paste even for amounts of 15 to 19.5% crosslinked gelatin sothat the hemostatic composition can be applied e.g. in microsurgery; on the other hand, theamounts shall be as low as to prevent negative functional properties of the hemostaticcomposition, for example adherence to wounds or hemostatic performance. For example, ifthe extrusion enhancer is albumin (which is specifically preferred, especially human serumalbumin), it must be provided in an amount of between 0.5 to 5.0% (w/w) (= weight ofextrusion enhancer per weight of final composition), preferably 1.0 to 5.0 % (w/w), preferably2.0 to 4.5% (w/w), more preferred 1.5 to 5.0% (w/w), especially preferred about 1.5% (w/w).
Another preferred class of extrusion enhancers according to the present invention arephospholipids, such as phosphatidylcholine and -serine, or complex mixtures such aslecithins or soy bean oils.
In another preferred embodiment the present invention provides a hemostaticcomposition comprising crosslinked gelatin in particulate form suitable for use in hemostasis,wherein the composition is present in paste form containing 16.0 to 19.5% (w/w), preferably16.5 to 19.5% (w/w 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more preferred 16.5 to19.0% (w/w) or 16.8 to 17.8% (w/w), especially preferred 16.5 to 17.5% (w/w), and whereinthe composition comprises an extrusion enhancer. Preferably the extrusion enhancer ishuman serum albumin.
In another preferred embodiment the present invention provides a hemostaticcomposition comprising crosslinked gelatin in particulate form suitable for use in hemostasis,wherein the composition is present in paste form containing 15.0 to 19.5% (w/w) crosslinkedgelatin, preferably 16.0 to 19.5% (w/w), 16.5 to 19.5% (w/w), 17.0 to 18.5% (w/w) or 17.5 to18.5% (w/w), more preferred 16.5 to 19.0% (w/w) or 16.8 to 17.8% (w/w), especially 16.5 to17.5% (w/w), and wherein the composition comprises an extrusion enhancer in aconcentration of more than 0,8% (w/w), preferably about 3,3% (w/w). Preferably theextrusion enhancer is human serum albumin, e.g. in the above mentioned concentrations.
The hemostatic compositions according to the present invention, especially the onesthat use albumin as extrusion enhancer, have specific advantages over the compositionsusing lower amounts of crosslinked gelatin (13 to 14.5%), especially they have an enhancedin vivo efficacy. It was unexpectedly revealed within the course of the present invention that aformulation with a higher gelatin particle concentration results in greater hemostaticperformance both in ex vivo test methods that use whole human blood and in pre-clinicalanimal experiments. The products according to the present invention enable a reducedsurgical approximation time and a faster time to hemostasis.
The compositions according to the present invention have a mean extrusion force(employing the test method described in example 1) of 40 N or below, preferably below 35 N,especially preferred below 20 N.
According to preferred embodiment of the present invention, the hemostaticcomposition comprises glutaraldehyde-crosslinked gelatin or genipin (Methyl (1R,2R,6S)hydroxy(hydroxymethyl)oxabicyclo[4.3.0]nona-4,8-dienecarboxylate)-crosslinkedgelatin, preferably type B gelatin, more preferably type B gelatin of hide origin.
Preferably, the crosslinked gelatin is present as granular material.
The hemostatic composition according to the present invention preferably comprisesa gelatin polymer which is especially a type B gelatin polymer. Type B gelatin has proven tobe specifically advantageous for use in hemostatic agents as the base treatment is highlyeffective in generating gelatin of appropriate properties and in mitigating risk of viral andzoonotic infection. A specifically preferred gelatin preparation can be prepared by processingyoung bovine corium with 2 N NaOH for about 1 hour at room temperature, neutralizing to pH7-8, and heating to 70°C. The corium is then fully solubilized to gelatin with 3-10% (w/w),preferably 7-10% (w/w) gelatin in solution. This solution can be cast, dried and ground toprovide gelatin type B powder.
Preferably, the gelatin has a Bloom strength of 200 to 400, especially a type B gelatinwith a Bloom strength of 200 to 400. Bloom is a test to measure the strength of gelatin. Thetest determines the weight (in grams) needed by a probe (normally with a diameter of 0.5inch) to deflect the surface of the gel 4 mm without breaking it. The result is expressed inBloom (grades). To perform the Bloom test on gelatin, a 6.67% gelatin solution is kept for 17-18 hours at 10°C prior to being tested.
The hemostatic composition according to the present invention preferably containsthe crosslinked gelatin in particulate form, especially as granular material. This granularmaterial can rapidly swell when exposed to a fluid (i.e. the diluent) and in this swollen form iscapable of contributing to a flowable paste that can be applied to a bleeding site. Accordingto a preferred embodiment, the crosslinked gelatin is provided from a dry crosslinked gelatin.
This dry crosslinked gelatin powder can be prepared to re-hydrate rapidly if contacted with apharmaceutically acceptable diluent. The gelatin granules, especially in the form of a gelatinpowder, preferably comprise relatively large particles, also referred to as fragments or sub-units, as described in W098/08550A and W02003/007845A. A preferred (median) particlesize will be the range from 10 to 1.000 pm, preferably from 200 to 800 |jm, but particle sizesoutside of this preferred range may find use in many circumstances.
Usually, the gelatin particles have a mean particle diameter ("mean particle diameter"is the median size as measured by laser diffractometry; "median size" (or mass medianparticle diameter) is the particle diameter that divides the frequency distribution in half; fiftypercent of the particles of a given preparation have a larger diameter, and fifty percent of theparticles have a smaller diameter) from 10 to 1000 |jm, preferably 50 to 700[jm, 200 to700|jm, 300 to 550pm, especially preferred 350 to 550|jm (median size). Although the termspowder and granular (or granulates) are sometimes used to distinguish separate classes ofmaterial, powders are defined herein as a special sub-class of granular materials. Inparticular, powders refer to those granular materials that have the finer grain sizes, and thattherefore have a greater tendency to form clumps when flowing. Granules include coarsergranular materials that do not tend to form clumps except when wet.
The present crosslinked gelatin in particulate form suitable for use in hemostasis mayinclude dimensionally isotropic or non-isotropic forms. For example, the crosslinked gelatin inthe kit according to the present invention may be granules or fibers; and may be present indiscontinuous structures, for example in powder forms.
The dry gelatin composition is liquid absorbing. For example, upon contact withliquids, e.g. aqueous solutions or suspensions (especially a buffer or blood) the crosslinkedgelatin takes up the liquid and will display a degree of swelling, depending on the extent ofhydration. The material preferably absorbs from at least 400 %, preferably about 500% toabout 2000%, especially from about 500% to about 1300% water or aqueous buffer byweight, corresponding to a nominal increase in diameter or width of an individual particle ofsubunit in the range from e.g. approximately 50% to approximately 500%, usually fromapproximately 50% to approximately 250%. For example, if the (dry) granular particles havea preferred size range of 0.01 mm to 1.5 mm, especially of 0.05 mm to 1 mm, the fullyhydrated composition (e.g. after administration on a wound or after contact with an aqueousbuffer solution) may have a size range of 0.05 mm to 3 mm, especially of 0.25 mm to 1.5The dry compositions will also display a significant "equilibrium swell" when exposedto an aqueous re-hydrating medium (= pharmaceutically acceptable diluent, also referred toas reconstitution medium). Preferably, the swell will be in the range from 400% to 1300%,preferably 400% to 1000%, more preferred 500% to 1100%, especially preferred from 500%to 900%, depending on its intended use. Such equilibrium swell may be controlled e.g. (for acrosslinked polymer) by varying the degree of cross-linking, which in turn is achieved byvarying the cross-linking conditions, such as the duration of exposure of a cross-linkingagent, concentration of a cross-linking agent, cross-linking temperature, and the like.
Materials having differing equilibrium swell values perform differently in different applications.
The ability to control crosslinking and equilibrium swell allows the compositions of thepresent invention to be optimized for a variety of uses. In addition to equilibrium swell, it isalso important to control the hydration of the material immediately prior to delivery to a targetsite. Hydration and equilibrium swell are, of course, intimately connected. A material with 0%hydration will be non-swollen. A material with 100% hydration will be at its equilibrium watercontent. Hydrations between 0% and 100% will correspond to swelling between the minimumand maximum amounts. "Equilibrium swell" may be determined by subtracting the dry weightof the gelatin hydrogel powder from its weight when fully hydrated and thus fully swelled. Thedifference is then divided by the dry weight and multiplied by 100 to give the measure ofswelling. The dry weight should be measured after exposure of the material to an elevatedtemperature for a time sufficient to remove substantially all residual moisture, e.g., two hoursat 120°C. The equilibrium hydration of the material can be achieved by immersing the drymaterial in a pharmaceutically acceptable diluent, such as aqueous saline, for a time periodsufficient for the water content to become constant, typically for from 18 to 24 hours at roomtemperature.
The crosslinked gelatin may be provided as a film which can then be milled to form agranular material. Most of the particles contained in a granular material (e.g. more than 90%w/w) have preferably particle sizes of 10 to 1.000|jm, preferably 50 to 700|jm, 200 to 700pm,300 to 550|jm, especially preferred 350 to 550|jm.
Preferably, the flowable form of the hemostatic composition contains particles that aremore than 50% (w/w) with a size of 100 to 1000 pm, preferably more than 80% (w/w) with asize of 100 to 1000pm.
Examples of suitable gelatin materials for crosslinking are described i.a. in examples1 and 2 of EP1803417B1 and example 14 of US6,066,325A and US6,063,061A. Gelatin mayalso be used with processing aids, such as PVP, PEG and/or dextran as re-hydration aids.
In one particular aspect of the present invention, compositions will comprisecrosslinked gelatin powders having a moisture content of 20% (w/w) or less, wherein thepowder was crosslinked in the presence of a re-hydration aid so that the powder has anaqueous re-hydration rate which is at least 5% higher than the re-hydration rate of a similarpowder prepared without the re-hydration aid. The "re-hydration rate" is defined according toEP1803417B1 to mean the quantity of an aqueous solution, typically 0.9% (w/w) saline thatis absorbed by a gram of the powder (dry weight basis) within thirty seconds, expressed asg/g. The rehydration rate is measured by mixing the crosslinked gelatin with saline solutionfor 30 seconds and depositing the wet gelatin on a filter membrane under vacuum to removethe free aqueous solution. One then records the weight of the wet gelatin retained on thefilter, dries it (e.g. 2 hr at 120°C), then records the dry weight of the gelatin and calculates theweight of solution that was absorbed per gram of dry gelatin.
Preferred compositions of the present invention will have a re-hydration rate of atleast 3 g/g, preferably at least 3.5 g/g, and often 3.75 g/g or higher. Re-hydration rates ofsimilar powders prepared without the re-hydration aids are typically below three, and apercentage increase in re-hydration rate will usually be at least 5%, preferably being at least%, and more preferably being at least 25% or higher.
Crosslinking can be done with any suitable crosslinker, e.g. glutaraldehyde such ase.g. described in W098/08550A and W02003/007845A. Crosslinking can also be carried outwith a non-toxic crosslinker such as genipin and the like.
Production cost is less for a genipin crosslinked gelatin product according to thepresent invention than a glutaraldehyde crosslinked one, since reagent, energy, and timecosts are lower. The genipin crosslinked gelatin reaction can be performed in water at neutralpH at room temperature for 5 16 hours. The product can be cleaned-up by an ethanol and/orwater wash which is not only cheaper, but more importantly, safer for the operator.
The method preferably applies the gelatin as being present in dry form before thecrosslinking step.
The preferred genipin-type crosslinker according to the present invention is, ofcourse, genipin (Methyl (^,2/?,6S)hydroxy(hydroxymethyl)oxabicyclo[4.3.0]nona-4,8-dienecarboxylate); however, also other crosslinkers of the iridoid- or secoiridoid-typemay be used, such as oleuropein. Preferred concentrations of genipin for crosslinking are inthe range of 0.5 to 20 mM, preferably 1 to 15 mM, especially 2 to 10 mM.
According to a preferred embodiment of the present invention, the genipin crosslinkedgelatin is subjected to a quenching/oxidation step with oxidizing agents such as bleach, tBu-hydroperoxide, etc., preferably to a treatment with sodium percarbonate, sodiumhypochlorite, chlorine water or hydrogen peroxide (N202), especially preferred is a treatmentwith sodium percarbonate or N262 most preferred is a treatment with percarbonate.
Preferred N202 concentrations are 0.5 to 20% (w/w), especially 1 to 15% (w/w), morepreferred about 5 % (w/w). In an especially preferred embodiment the genipin concentrationis between 5 to 10 mM, the reaction time of gelatin with genipin is between 3 to 10 hours,especially 6 hours, the HzOz concentration is between 3 to 5 %(w/w) and the reaction time ofthe genipin-crosslinked gelatin with N202 is about 20 hours,Preferred percarbonate concentrations are between 1 to 10 % (w/w), especially 1 to 5% (w/w), more preferred 1 to 4 % (w/w). In an especially preferred embodiment the genipinconcentration is between 5 to 10 mM (especially about 8 nM), the reaction time of gelatinwith genipin is between 3 to 10 hours (especially about 5 hours), the percarbonateconcentration is between 1 to 10 % (w/w), especially preferred between 1 to 4 % w/w, andthe reaction time of the genipin-crosslinked gelatin with percarbonate is between 1 to 20hpurs, preferably between 1 to 5 hours (e.g. 1, 2 or 3 hours).
Quenching may also be carried out in presence of antioxidants such as sodium ascorbate orby controlling oxidation potential of the reaction environment such as carrying out quenchingand/or genipin reaction in an inert atmosphere such as nitrogen or argon.
Preferred crosslinking reaction conditions include the performance in aqueoussolution, preferably in a phosphate buffered saline (PBS)/ethanol buffer, especially at a pH of4 to 12, preferably of 5.0 to 10.0, especially of 6 to 8, or in deionized water or other aqueousbuffers which may contain between 0 to 50% of a water miscible organic solvent. A PBSbuffer contains physiological amounts of NaCI and KCI in a phosphate buffer at aphysiological pH. An example for a PBS buffer contains 137 mM NaCI, 2.7 mM KCI, 10 mMNa2HP04 • 2 H20, 1.76 mM KH2P04 (pH = 7.4). Another example of a PBS buffer consists of137 mM NaCI, 2.7 mM KCI, 4.3 mM Na2HP04 and 1.4 mM KH2P04 (pH = 7.5).
The reaction may also be carried out in an aqueous buffer containing up to 50% of awater-miscible organic solvent and/or processing aids such as PEG, PVP, mannitol, sodiumpercarbonate, sodium lactate, sodium citrate, sodium ascorbate etc..
Preferably, the crosslinking step is performed at a temperature of 4°C to 45°C,preferably of 15 to 45°C, especially of 20 to 40°C.
The crosslinking step may be followed by a quenching step, especially with an amino-group containing quencher, preferably an amino acid, especially glycine. With the quencher,yet unreacted genipin-type crosslinkers are inactivated (e.g. by reaction with the quencher inexcess) to prevent further crosslinking. Quenching may also be carried out by raising pH ofsolution to between 8 to 14, or by using nucleophilic compounds containing amino, thiol, orhydroxyl groups and also a combination of pH raising and using nucelophilic compounds.
The quenching step after the genipin-gelatin crosslinking reaction according to the presentinvention can be actively directed to impart desired physical performance such as swell andTEG which are important determinants of hemostatic activity above and beyond the generalgenipin-crosslinking alone.
The crosslinked gelatin is preferably washed after the crosslinking step, preferably bymethanol, ethanol or water, especially by deionized water. Another preferred washing stepapplies an aqueous buffer containing up to 50% (v/v) of water-miscible organic solventand/or one or more processing aids.
According to a preferred embodiment, the crosslinked gelatin is dried. In such a driedstate, the hemostatic composition is storage-stable for long time even at elevatedtemperatures (e.g. more than 20°C, more than 30°C or even more than 40°C). Preferreddryness conditions include crosslinked biocompatible polymers which are dried to have amoisture content of below 15% (w/w), preferably below 10%, more preferred below 5%,especially below 1%. In another preferred embodiment the product may be supplied in ahydrated or wet state where the hydrating solution may be a biocompatible buffer or solution.
A Glu-Gel product has a tendency to be camouflaged by surrounding tissue, since it's slightlyyellow color blends in with it. This makes visual evaluation of the desired applicationproblematic. The genipin crosslinked gelatin products according to the present inventionappear variable color from pale yellow to dark blue or green based upon degree ofcrosslinking reaction conditions, and subsequent processing and finishing steps. Thistunability of color and ability to obtain desired color in finished product color has the addedadvantage of providing physicians visual indication of proper product application in woundsites, since this color differentiates it from surrounding tissue, instead of potentially beingcamouflaged by it. This is another novel feature of this invention. On the other hand, thecolor can be removed to obtain a non-colored product, depending on the needs with respectto the final products.
In a preferred embodiment the biocompatible polymer, e.g. gelatin, crosslinked with agenipin-type crosslinker, e.g. genipin, is a homogeneously (uniformely) crosslinked polymeras can be shown e.g. by fluorescence measurements as described in Example 3 of thepresent application. In an especially preferred embodiment the biocompatible polymer, suchas gelatin, is present as a homogeneously genipin crosslinked biocompatible polymer, suchas gelatin, in particulate form.
A hemostatic composition according to the present invention is preferred, whereinexcipients, such as lubricants, e.g. hyaluronic acid, are present.
In another embodiment of the present invention excipients, such as lubricants, e.g.hyaluronic acid, are excluded.
The pharmaceutically acceptable diluent is preferably an aqueous solution and maycontain a substance selected from the group consisting of NaCI, CaCl2 and sodium acetate.
For example, a pharmaceutically acceptable diluent comprises water for injection, and -independently of each other - 50 to 200 mM NaCI (preferably 150 mM), 10 to 80 mM CaCl2(preferably 40 mM) and 1 to 50 mM sodium acetate (preferably 20 mM). In anotherembodiment the pharmaceutically acceptable diluent contains less than 35 g/1 of mannitol,preferably less than 25 g/1, more preferred less than 10 g/l, especially preferred thepharmaceutically acceptable diluent is essentially free of mannitol.
According to a preferred embodiment, the pharmaceutically acceptable diluentcomprises thrombin, preferably 10 to 1000 I.U. thrombin/ml, especially 250 to 700 I.U.thrombin/ml. Preferably, the hemostatic composition in this ready to use form contains 10 to100.000 International Units (I.U.) of thrombin, more preferred 100 to 10.000 I.U., especially500 to 5.000 I.U.. Thrombin (or any other coagulation inducing agent, such as snake venom,a platelet activator, a thrombin receptor activating peptide and a fibrinogen precipitatingagent) can be derived from any thrombin preparation which is suitable for use in humans (i.e.pharmaceutically acceptable). Suitable sources ofthrombin include human and bovine blood,plasma or serum (thrombin of other animal sources can be applied if no adverse immunereactions are anticipated), thrombin of recombinant origin (e.g. human recombinant thrombin)and autologous human thrombin can be preferred for some applications.
The pharmaceutically acceptable diluent is used in an amount to achieve the desiredend-concentration in the ready-to-use composition. The thrombin preparation may containother useful component, such as ions, buffers, excipients, stabilizers, etc.. Preferably, thethrombin preparation contains human albumin as the extrusion enhancer. Preferred salts areNaCI and/or CaClg, both used in the usual amounts and concentrations applied for thrombin(e.g. 0.5 to 1.5 % NaCI (e.g. 0.9%) and/or 20 to 80 mM CaClz (e.g. 40 mM)).
In a further embodiment, the diluent can also include a buffer or buffer system so as to bufferthe pH of the reconstituted dry composition, preferably at a pH of 3.0 to 10.0, more preferredof 6.4 to 7.5, especially at a pH of 6.9 to 7.1.
Establishment of appropriate amounts of crosslinked gelatin, diluent and extrusionenhancer may be made in the kit according to the aforementioned prerequisites: Forexample a) a vial with 0.736 to 0,995 g dry crosslinked gelatin (corresponding to 15.0 to19.5% (w/w) in the final product) may be provided and b) a second vial with 4 ml diluent with60 to 240 mg albumin and, optionally, thrombin at a concentration of 500 I.U./ml and/or 40mM CaCla. Alternatively, albumin may be added in lyophilized form to the dry gelatincomponent a) of the kit. For example, a) a vial with 0.573 to 0.775 g dry crosslinked gelatin(corresponding to 15.0 to 19.5% (w/w) in the final product) thereof 48 to 192 mg albumin maybe provided and b) a second vial with 3.2 ml diluent and, optionally, thrombin at aconcentration of 500 I.U./ml and/or 40 mM CaCl2.
The crosslinked gelatin component of the kit according to the present invention ispreferably provided as a dry composition, wherein the crosslinked gelatin is present in dryform.
A substantially dry crosslinked gelatin composition according to the present inventionhas a residual content of moisture which may approximately correspond to the moisturecontent of comparable available products, such as Floseal® (Floseal, for example, hasapproximately 8-12% moisture as a dry product).
The dry crosslinked gelatin in particulate form suitable for use in hemostasis in the kitaccording to the present invention is preferably gelatin in powder form, especially whereinthe powder particles have a median particle size of 10 to 1000|jm, preferably 50 to 700pm,200 to 700pm, 300 to 550|jm, especially preferred 350 to 550pm. A "dry granular preparationof crosslinked gelatin" according to the present invention is in principle known e.g. fromW098/08550A. Preferably, the crosslinked gelatin is a biocompatible, biodegradable drystable granular material.
According to another aspect, the present invention relates to a hemostaticcomposition according to the present invention for use in the treatment of an injury selectedfrom the group consisting of a wound, a hemorrhage, damaged tissue, bleeding tissue and/orbone defects.
Another aspect of the present invention is a method of treating an injury selected fromthe group consisting of a wound, a hemorrhage, damaged tissue and/or bleeding tissuecomprising administering a hemostatic composition according to the present invention to thesite of injury.
According to another aspect, the present invention also provides a method fordelivering a hemostatic composition according to the invention to a target site in a patient'sbody, said method comprising delivering a hemostatic composition produced by the processaccording to the present invention to the target site. Although also the dry composition canbe directly applied to the target site (and, optionally be contacted with a diluent at the targetsite, if necessary), it is preferred to contact the dry hemostatic composition with apharmaceutically acceptable diluent before administration to the target site, so as to obtain ahemostatic composition according to the present invention in paste form.
In such a method, a kit for making a flowable paste of crosslinked gelatin for thetreatment of an injury selected from the group consisting of a wound, a hemorrhage,damaged tissue and/or bleeding tissue, may be applied, this kit comprisinga) a dry hemostatic composition comprising crosslinked gelatin in particulate form to bereconstituted to a flowable paste containing 15.0 to 19.5% (w/w) crosslinked gelatin (= weightof dry gelatin to weight of final composition), preferably 16.0 to 19.5% (w/w), 16.5 to 19.5%(w/w), 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more preferred 16.5 to 19.0% (w/w) or16.8 to 17.8% (w/w), especially preferred 16.5 to 17.5% (w/w), crosslinked gelatin andb) a pharmaceutically acceptable diluent for reconstitution of the hemostatic composition,wherein either the composition or the diluent comprises an extrusion enhancer, especiallyalbumin, in a suitable amount, for example (for albumin) in an amount which leads to analbumin concentration in the reconstituted paste of between 0.5 to 5.0% (w/w) (= weight ofextrusion enhancer per weight of final composition), preferably 1.0 to 5.0 % (w/w), preferably2.0 to 4.5% (w/w), more preferred 1.5 to 5.0% (w/w), especially preferred about 1.5% (w/w).
A preferred further component of such a kit is - specifically if the hemostaticcomposition is contained in dry form - a diluent for reconstitution (= re-hydration medium) ofthe hemostatic composition. Further components of the kit may be administration means,such as syringes, catheters, brushes, etc. (if the compositions are not already provided in theadministration means) or other components necessary for use in medical (surgical) practice,such as substitute needles or catheters, extra vials or further wound cover means.
Preferably, the kit according to the present invention comprises a syringe housing the dryand stable hemostatic composition and a syringe containing the diluent (or provided to takeup the diluent from another diluent container).
In a preferred embodiment, the pharmaceutically acceptable diluent is provided in aseparate container. This can preferably be a syringe. The diluent in the syringe can theneasily be applied to the final container for reconstitution of the dry hemostatic compositionsaccording to the present invention. If the final container is also a syringe, both syringes canbe finished together in a pack. It is therefore preferred to provide the dry hemostaticcompositions according to the present invention in a syringe which is finished with a diluentsyringe with a pharmaceutically acceptable diluent for reconstituting said dry and stablehemostatic composition.
According to a preferred embodiment, the final container further contains an amountof a stabilizer effective to inhibit modification of the polymer when exposed to the sterilizingradiation, preferably ascorbic acid, sodium ascorbate, other salts of ascorbic acid, or anantioxidant.
With such a pharmaceutically acceptable diluent, a ready to use form of the presenthemostatic composition may be provided which can then be directly applied to the patient.
Accordingly, also method for providing a ready to use form of a hemostatic compositionaccording to the present invention is provided, wherein the hemostatic composition isprovided in a first syringe and a diluent for reconstitution is provided in a second syringe, thefirst and the second syringe are connected to each other, and the fluid is brought into the firstsyringe to produce a flowable form of the hemostatic composition; and optionally returningthe flowable form of the hemostatic composition to the second syringe at least once.
Preferably, the ready-to use preparations are present or provided as hydrogels. Products ofthis kind are known in principle in the art, yet in a different format. Therefore, a method forproviding a ready to use form of a hemostatic composition according to the present invention,wherein the hemostatic composition is provided in a first syringe and a diluent forreconstitution is provided in a second syringe, the first and the second syringe are connectedto each other, and the diluent is brought into the first syringe to produce a flowable form ofthe hemostatic composition; and optionally returning the flowable form of the hemostaticcomposition to the second syringe at least once, is a preferred embodiment of the presentinvention. This process (also referred to as "swooshing") provides a suitable ready-to-useform of the compositions according to the present invention which can easily and efficientlybe made also within short times, e.g. in emergency situations during surgery. This flowableform of the hemostatic composition provided by such a method is specifically suitable for usein the treatment of an injury selected from the group consisting of a wound, a hemorrhage,damaged tissue, bleeding tissue and/or bone defects.
For stability reasons, such products (as well as the products according to the presentinvention) are usually provided in a dry form in a final container and brought into the ready-to-use form (which is usually in the form of a (hydro)gel, suspension or solution) immediatelybefore use, necessitating the addition of a pharmaceutically acceptable diluents (= re-hydration medium).
According to another aspect, the present invention relates to a method for providing aready to use form of a hemostatic composition according to the present invention, whereinthe hemostatic composition is provided in a first syringe and a diluent for reconstitution isprovided in a second syringe, the first and the second syringe are connected to each other,and the fluid is brought into the first syringe to produce a flowable form of the hemostaticcomposition; and optionally returning the flowable form of the hemostatic composition to thesecond syringe at least once.
Preferably, the flowable form of the hemostatic composition according to the presentinvention contains more than 50% (w/w) particles with a size of 100 to 1000 pm, preferablymore than 80% (w/w) particles with a size of 100 to 1000 pm.
The biocompatible hemostatic crosslinked polymer according to the present invention- once applied to a wound - forms an efficient matrix which can form a barrier for blood flow.
Specifically the swelling properties of the hemostatic polymer can make it an effectivemechanical barrier against bleeding and re-bleeding processes.
The present composition may additionally contain a hydrophilic polymeric component(also referred to as "reactive hydrophilic component" or "hydrophilic (polymeric) crosslinker")which further enhances the adhesive properties of the present composition. This hydrophilicpolymeric component of the haemostatic composition according to the present invention actsas a hydrophilic crosslinker which is able to react with its reactive groups once thehaemostatic composition is applied to a patient (e.g. to a wound of a patient or another placewhere the patient is in need of a hemostatic activity). Therefore it is important for the presentinvention that the reactive groups of the hydrophilic polymeric component are reactive whenapplied to the patient. It is therefore necessary to manufacture the haemostatic compositionaccording to the present invention so that the reactive groups of the polymeric componentwhich should react once they are applied to a wound are retained during the manufacturingprocess.
For hydrophilic polymeric crosslinkers whose reactive groups are hydrolysable,premature contact with water or aqueous liquids has to be prevented before administration ofthe haemostatic composition to the patient, especially during manufacture. However,processing of the hydrophilic polymeric component during manufacturing may be possiblealso in an aqueous medium at conditions where the reactions of the reactive groups areinhibited (e.g. at a low pH). If the hydrophilic polymeric components can be melted, themelted hydrophilic polymeric components can be sprayed or printed onto the matrix ofcrosslinked gelatin. It is also possible to mix a dry form (e.g. a powder) of the hydrophilicpolymeric component with a dry form of the crosslinked gelatin. If necessary, then anincrease of the temperature can be applied to melt the sprinkled hydrophilic polymericcomponent to the crosslinked gelatin to achieve a permanent coating of the haemostaticcomposition. Alternatively, these hydrophilic polymeric components can be taken up into inertorganic solvents (inert vis-a-vis the reactive groups of the hydrophilic polymeric components)and brought onto the matrix of the crosslinked gelatin. Examples of such organic solvents aredry ethanol, dry acetone or dry dichloromethane (which are e.g. inert for hydrophilicpolymeric components, such as NHS-ester substituted PEGs). Alternatively, nucleophilicgroups may also be added (e.g. PEG-SH).
In a preferred embodiment the hydrophilic polymer component is a single hydrophilicpolymer component and is a polyalkylene oxide polymer, preferably a PEG comprisingpolymer. The reactive groups of this reactive polymer are preferably electrophilic groups.
The reactive hydrophilic component may be a multi-electrophilic polyalkylene oxidepolymer, e.g. a multi-electrophilic PEG. The reactive hydrophilic component can include twoor more electrophilic groups, preferably a PEG comprising two or more reactive groupsselected from succinimidylesters (-CON(COCH2)2), aldehydes (-CHO) and isocyanates (-N=C=0), e.g. a component as disclosed in the W02008/016983 A (incorporated herein byreference in its entirety).
Preferred electrophilic groups of the hydrophilic polymeric crosslinker according to thepresent invention are groups reactive to the amino-, carboxy-, thiol- and hydroxy- groups ofproteins, or mixtures thereof.
Preferred amino group-specific reactive groups are NHS-ester groups, imidoestergroups, aldehyde-groups, carboxy-groups in the presence of carbodiimides, isocyanates, orTHPP (beta-[Tris(hydroxymethyl)phosphino] propionic acid), especially preferred isPentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate (= Pentaerythritoltetrakis[1-1'-oxo-5'-succinimidylpentanoatepoly-oxoethyleneglycole]ether (= an NHS-PEGwith MW 10,000).
Preferred carboxy-group specific reactive groups are amino-groups in the presence ofcarbodiimides.
Preferred thiol group-specific reactive groups are maleimides or haloacetyls.
Preferred hydroxy group-specific reactive group is the isocyanate group.
The reactive groups on the hydrophilic cross-linker may be identical (homo-functional) ordifferent (hetero-functional). The hydrophilic polymeric component can have two reactivegroups (homo-bifunctional or hetero-bifunctional) or more (homo/hetero-trifunctional ormore).
In special embodiments the material is a synthetic polymer, preferably comprisingPEG. The polymer can be a derivative of PEG comprising active side groups suitable forcross-linking and adherence to a tissue.
By the reactive groups the hydrophilic reactive polymer has the ability to cross-linkblood proteins and also tissue surface proteins. Cross-linking to the crosslinked gelatin isalso possible,The multi-electrophilic polyalkylene oxide may include two or more succinimidylgroups. The multi-electrophilic polyalkylene oxide may include two or more maleimidylgroups.
Preferably, the multi-electrophilic polyalkylene oxide is a polyethylene glycol or aderivative thereof.
In a most preferred embodiment the hydrophilic polymeric component ispentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate (=COH102, alsopentaerythritoltetrakis[1-1'-oxo-5'-succinimidylpentanoatepoly-oxoethyleneglycole]ether).
The hydrophilic polymeric component is a hydrophilic crosslinker. According to apreferred embodiment, this crosslinker has more than two reactive groups for crosslinking("arms"), for example three, four, five, six, seven, eight, or more arms with reactive groups forcrosslinking. For example, NHS-PEG-NHS is an effective hydrophilic crosslinker according tothe present invention. However, for some embodiments, a 4-arm polymer (e.g. 4-arms-p-NP-PEG) may be more preferred; based on the same rationale, an 8-arm polymer (e.g. 8-arms-NHS-PEG) may even be more preferred for those embodiments where multi-reactivecrosslinking is beneficial. Moreover, the hydrophilic crosslinker is a polymer, i.e. a largemolecule (macromolecule) composed of repeating structural units which are typicallyconnected by covalent chemical bonds. The hydrophilic polymer component should have amolecular weight of at least 1000 Da (to properly serve as crosslinker in the hemostaticcomposition according to the present invention); preferably the crosslinking polymersaccording to the present invention has a molecular weight of at least 5000 Da, especially ofat least 8000 Da.
For some hydrophilic crosslinkers, the presence of basic reaction conditions (e.g. atthe administration site) is preferred or necessary for functional performance (e.g. for a fastercross-linking reaction at the administration site). For example, carbonate or bicarbonate ions(e.g. as a buffer with a pH of 7.6 or above, preferably of 8.0 or above, especially of 8.3 andabove) may be additionally provided at the site of administration (e.g. as a buffer solution oras a fabric or pad soaked with such a buffer), so as to allow an improved performance of thehemostatic composition according to the present invention or to allow efficient use as ahemostatic and/or wound adherent material.
The reactivity of the hydrophilic polymeric component (which, as mentioned, acts as acrosslinker) in the composition according to the present invention is retained in thecomposition. This means that the reactive groups of the crosslinker have not yet reacted withthe haemostatic composition and are not hydrolyzed by water (or at least not in a significantamount which has negative consequences on the hemostatic functionality of the presentcompositions). This can be achieved by combining the crosslinked gelatin with thehydrophilic crosslinker in a way which does not lead to reaction of the reactive groups of thecrosslinker with the hemostatic polymer or with water. Usually, this includes the omitting ofaqueous conditions (or wetting), especially wetting without the presence of acidic conditions(if crosslinkers are not reactive under acidic conditions). This allows the provision of reactivehaemostatic materials.
Preferred ratios of the crosslinked gelatin to hydrophilic polymeric component in thehemostatic composition according to the present invention are from 0.1 to 50% (w/w),preferably from 5 to 40% (w/w).
Further components may be present in the hemostatic composition according to thepresent invention. According to preferred embodiments, the hemostatic compositionsaccording to the present invention may further comprise a substance selected from the groupconsisting of antifibrinolytic, procoagulant, platelet activator, antibiotic, vasoconstrictor, dye,growth factors, bone morphogenetic proteins and pain killers.
The present invention also refers to a finished final container containing thehemostatic composition according to the present invention. This finished container containsthe hemostatic composition according to the present invention in a sterile, storage-stable andmarketable form. The final container can be any container suitable for housing (and storing)pharmaceutically administrable compounds. Syringes, vials, tubes, etc. can be used;however, providing the hemostatic compositions according to the present invention in asyringe is specifically preferred. Syringes have been a preferred administration means forhemostatic compositions as disclosed in the prior art also because of the handlingadvantages of syringes in medical practice. The compositions may then preferably beapplied (after reconstitution) via specific needles of the syringe or via suitable catheters. Thereconstituted hemostatic compositions (which are preferably reconstituted to form ahydrogel) may also be applied by various other means e.g. by a spatula, a brush, a spray,manually by pressure, or by any other conventional technique. Administration of thereconstituted hemostatic composition to a patient by endoscopic (laparoscopic) means isspecifically preferred. Usually, the reconstituted hemostatic compositions according to thepresent invention will be applied using a syringe or similar applicator capable of extruding thereconstituted composition through an orifice, aperture, needle, tube, or other passage to forma bead, layer, or similar portion of material. Mechanical disruption of the compositions can beperformed by extrusion through an orifice in the syringe or other applicator, typically having asize in the range from 0.01 mm to 5.0 mm, preferably 0.5 mm to 2.5 mm. Preferably,however, the hemostatic composition will be initially prepared from a dry form having adesired particle size (which upon reconstitution, especially by hydration, yields subunits ofthe requisite size (e.g. hydrogel subunits)) or will be partially or entirely mechanicallydisrupted to the requisite size prior to a final extrusion or other application step. It is, ofcourse evident, that these mechanical components have to be provided in sterile form (insideand outside) in order to fulfill safety requirements for human use.
The hemostatic composition according to the present invention is preferably appliedin its pasty form to a patient from a container as described in Example 1 with an extrusionforce of 40 N or lower, such as lower SON or lower 20N, preferably in a range of 15 to 30 N.
Another aspect of the invention concerns a method for providing a ready-to-usehemostatic composition comprising contacting a hemostatic composition according to thepresent invention.
The invention is further described in the examples below and the drawing figures, yetwithout being restricted thereto.
Figure 1 shows the mean extrusion force of glutaraldehyde crosslinked gelatin pastescontaining 17.5% (w/w) crosslinked gelatin with various concentrations of human serumalbumin in the thrombin component (extrusion force needed to push product out of syringe atcompression speed 250mm/min, calculated at 35 mm distance; all products incubated for 30minutes at room temperature, quick re-swooshing shortly before extrusion forcemeasurement). The x-axis shows the human serum albumin concentration in the thrombincomponent in [g/1], the y-axis shows the mean extrusion force in [N].
Figure 2 shows the consistency of crosslinked gelatin pastes containing 17.5% (w/w)crosslinked gelatin depending on the concentration of human serum albumin.
Figure 3 shows the mean extrusion force of genipin crosslinked gelatin pastescontaining 17.5% (w/w) gelatin with various concentrations of human serum albumin in thethrombin component. The x-axis shows the human serum albumin concentration in thethrombin component in [g/1], the y-axis shows the mean extrusion force in [N].
Figure 4 shows evaluation of bleeding severity post test article application andapproximation.
Figures 5 to 8 show the hemostatic efficacy in Porcine Liver Punch-Biopsy Model ofdifferent preparations. The x-axis shows the time after application in [seconds], the y-axisshows percent of hemostatic success (defined as "no bleeding" in Figure 5 and as "nobleeding" or "ooze" in Figure 6).
In Figures 5 and 6 the symbols mean:glutaraldehyde crosslinked gelatin with 50 g/1 human serum albumin in the thrombinsolution (n=8)glutaraldehyde crosslinked gelatin with 75 g/1 human serum albumin in the thrombinsolution (n=8)In Figures 7 and 8 the symbols mean:, = 17.5% (w/w) glutaraldehyde crosslinked gelatins 14.5% (w/w) glutaraldehyde crosslinked gelatin" 17.5%(w/w) glutaraldehyde crosslinked gelatin plus 2.5% PEG10.000 in thrombinsolutionEXAMPLESExample 1: Determination ofExtrusion Force (EF):An Instron model 5544 mechanical tester equipped with a 100 N load cell operating at across-beam speed of 250 mm/min was used to measure extrusion forces needed to extrudethe product from a syringe. The necessary extrusion forces were measured during thecomplete cross-beam displacement (34mm deflection) which corresponds to a distance asyringe plunger moves in order to extrude almost the entire product out of the syringe. Fromthese forces the mean extrusion forces were calculated as follows:Total Enemy OnJ) _ = Mean Force (N)Max. Deflection (mm)Samples for this test were prepared as follows: A 5 ml standard syringe (with a cylindric bodyhaving an inner diameter of 12.2mm) with a male luer lock system (the inner nozzle lumendiameter where the adapter is attached measures 2.54mm) is filled with 0.704 g dry mass ofthe solid sample (approx. 0.8g taking the residual moisture of approx. 12% into account). Asa diluent 3.2 ml of a thrombin solution containing 5001U/ml thrombin in 40mM calciumchloride and either 0, 5, 15, 25, 50 or 75 mg/ml human serum albumin was used. The diluentand the solid component were mixed by connecting the syringe holding the diluent (astandard 5ml syringe with a female luer lock system) and the syringe holding the drycomponent and pushing the contents back and forth at least 10 times (this mixing techniqueis called "swooshing"). Thereafter the sample was incubated for 30 min at room temperaturebefore measurement. After incubation each sample was "re-swooshed" two times and thesyringe holding the product (the syringe that previously held the dry component asmentioned above) was connected to a malleable applicator (female luer connector system,inner tube diameter of 2.29mm holding two wires and having a total length of 141.5mm). Thesyringe was assembled to the applicator and placed into the Instron set up and the test wasstarted.
The syringes and the applicator were commercially available as parts of the FlosealHemostatic Matrix product from Baxter.
The results for a glutaraldehyde crosslinked gelatin as in Floseal are depicted in Fig.1 and those for a genipin crosslinked gelatin as described below are depicted in Fig. 3 andalso shown as corresponding Table 1 and Table 2.
The consistency of crosslinked gelatin pastes containing 17.5% (w/w) crosslinked gelatindepending on the concentration of albumin is shown in Fig. 2 (with 0, 25, 50 and 75 g/1human serum albumin provided in the thrombin component).
Table 1:extrusion force stdc(albumin) [g/1] in thethrombin component dev_[N]_0 4038 1,53025 2,250 1960 1,0Fable 2:c(albumin) g/1 in the extrusion forcethrombin component dev29Preparation ofgenipin crosslinked gelatin:Bovine derived collagen was processed via alkaline treatment and subsequently rinsed withdeionized process water (DIW) to remove residual salts. Gelatin was extracted by heattreatment and dried in sheets. The sheets were ground to a powder that was to be processedusing genipin as a crosslinking agent.1kg of gelatin granules were added to 201 of a 10mM genipin solution in DIW. The reactionwas performed at neutral pH (7.2) in a jacketed temperature controlled tank at 23°C. Mixingwas carried out for 6 hours and the solution was drained off, retaining the solids within amesh, and rinsed through with DIW to wash out remaining genipin. The material was re-suspended in a 5% N202 solution for 20 hours. The material was rinsed through with DIW toremove the HzOs. The solids were pre-dried on filter paper under vacuum and then ovendried for 2.5 days. The dried matrix was ground to a powder and filled into individual plasticsyringes before exposure to gamma irradiation.
Example 2: Determination ofhemostatic efficiencyMaterials and Methods:Animal ModelFor this model, a midline laparotomy is performed, followed by electrocautery to stopthe bleeding from the surgical incision. The liver is exposed and a lobe is isolated. A 10 mmdiameter punch biopsy is used to create a series of 2, non-full thickness lesions,approximately 5 mm deep, with the core tissue removed. A pre-treatment assessment ismade on the lesion which includes collecting the blood flowing from each lesion for 10 sec.with pre-weighed gauze.
Test articles are randomized and presented to the surgeon who is blinded to thesample treatment. Approximately 1.0 ml of the assigned test article is topically applied to alesion. Saline moistened gauze is used to help approximate the test articles to theirdesignated lesions, and the timer is started. The saline moistened approximation gauze isremoved after 30 seconds.
The degree of bleeding is assessed at 30, 60, 90, 120, 300, and 600 see. after thetest articles are applied to their designated lesions as per the depictions in fig. 3 (Bleedingscore: 0: no bleeding (product saturated with blood); 1: ooze (blood out of product but noblood drop); 2: very mild (blood drop on the product); 3: mild (blood drop streams down); 4:moderate (small amount of blood streams down); 5: severe (large amount of blood streamsdown)). .
Product saturated with blood, but without active bleeding is scored as a "0" (zero).
Saline is used to irrigate the excess test articles away from the lesions after the 300 sec.assessment. The procedure is repeated and performed in multiple liver lobes. A singlesurgeon creates, treats, and performs the observation assessments.
Test Article SynthesisTest articles for the in vivo evaluation in the porcine-liver model were made bypreparing pastes of crosslinked gelatin (in concentrations of 14.5% and 17.5% with 25 or 50g/1 human serum albumin in the thrombin solution (with or without additional 2.5% PEG)).
The results are depicted in Figs. 5 to 8 showing improved performance with 17.5 %gelatin and less effectiveness in the presence of plasticizers (PEG).
Example 3:Gelatin samples were formulated per the package insert for Floseal with a couple keyexceptions. First, sodium chloride was used instead of calcium chloride and the gelatin wasformulated at 125% solids instead of 100%. The gelatin/thrombin formulations were allowedto stand for 25 minutes and then 1 ml of the preparation was discarded. The other 1ml ofmaterial was applied to the topical hemostasis system (THS). The THS apparatus waspreviously primed with platelet poor plasma.
The THS is an apparatus designed to simulate a bleeding wound. The artificial wound is acylindrical hole in a silicone substrate. The surface of the silicone cylinder was coated with alayer of fibrinogen. A syringe pump expelled the clotting fluid (whole blood, plasma, etc.) inthis case platelet poor plasma, while the back pressure was recorded. In this experiment theplasma was flowed at a fixed rate of 0.25ml/min through a small hole at the bottom center ofthe cylindrical wound. The excess plasma was soaked up with gauze immediately prior toapplication of the hemostatic matrix. As the plasma continued to flow, 1ml of the hemostaticmatrix was applied to the cylindrical wound. This was immediately covered with wet gauzeand a fixed pressure was applied. After 30 seconds the weight was removed and the plasmacontinued to flow for 8-10 minutes, at which point the flow was stopped and the clot set asidein a humidity chamber where it stayed for more than 2 hours. At the end of the two hours, theclot was mounted onto a vibratome at 8°C, where approximately 500 pm thick slabs weresectioned from the clot. These sections were immersed into a PBS buffer. The slabs werestored in a 5°C refrigerator when not in use. The slab was placed onto a coverslip andimaged with a Nikon A1R confocal microscope running the NIS-Elements AdvancedResearch v3.22.00 Build 710 software. To collect micrographs, a plan fluor 10x objective wasused with laser excitation light at 488 nm and an emission collection window from 500-550nm. A transmitted light image was simultaneously collected using a transmitted light detector.
With these imaging parameters, automated stitching performed by the software was used togenerate macroscopic maps of samples. Smaller areas of the samples were alsocharacterized by collecting 3D z-stacks of images with an optical slice thickness of 5.125 [jm.
The composite confocal map was used to identify the gelatin granules that are located at thesurface, and which were sectioned. This was important for positioning of the elasticitymeasurement in the atomic force microscope (AFM). The clot slab was mounted in a VeecoMultimode AFM. The multimode was equipped with a Nanoscope V controller and a JVpiezoelectric scanner. The force measurements were made with a Novascan AFM cantieverwhich supported a 4.5 pm polystyrene sphere. The cantilever's force constant wasdetermined to be 0.779 N/m by the thermal tune method. The cantilever was positionedabove the center of the gelatin granule, and then a 16x16 array of force measurements weremade. Each force curve involved moving the gelatin granule up into contact with thepolystyrene sphere, and continuing to move the granule up until the cantilever deflectionreached a preset trigger value of 2 volts, at which point the gelatin was retracted a distanceof 1.00 micron from the trigger location.
DiscussionThe fluorescence data shows that the glutaraldehyde crosslinked gelatin is not uniformlycrosslinked. Instead, the crosslinking density seems higher around the edges of thegranules, with the central portion of the granule being significantly less crosslinked than theedges. In contrast, the genipin crosslinked gelatin appears uniformly (homogeneously)crosslinked throughout the granules. There are no substantial edge effects to thefluorescence intensity. The fluorescence intensity of the genipin and glutaraldehydecrosslinked materials cannot be directly compared, because of the potential fluorescencedifferences attributed to the crosslinkers themselves. However, the AFM measured elasticmodulus measurement show that the genipin crosslinked gelatin is stiffer than theglutaraldehyde crosslinked gelatin, which appears to be softer (more flexible).