REGENERATED OXIDIZED CELULOSE BASED HEMOSTATIC MATERIALCONTAINING ANTIFIBROLYTIC AGENTS

Abstract

In present invention oxidation method of viscon cellulose with NO.sub.2 obtained H.sub.3PO.sub.4/HNO.sub.3 is defined liquid and gas media. Regenerated oxidise cellulose (REOC in shorten term) contain in COOH yields are standardised as 18.6-20.1 for textile, 19.8-21.5% for powder samples. Powder and textile (woven and fabric) products are impregnated 1.8-2.4% Ca.sup.+2 ion, 0-1.1% Na.sup.+1 ion, 0.8-1.5% tranexamic acid and 6-aminocaproic acid as antifibrinolytic. Obtained powder and gel products are impregnated Bi.sup.+3, Zn.sup.+2 and Ag.sup.+1 ions for antiseptic purposes. Only Bismuth of them is shown antibacterial effects. Also the aim of present invention is haemostat antimicrobial properties during impregnation of Rifampicin, Gatifloxacin, Doxycycline, Levofloxacin, Lincomycin, Clindamycin, Ciprofloxacin. Haemostatic properties are indicated for all products and antimicrobial properties are shown for some samples. Cytotoxicity, sensitivity and irritation properties are determined in compliance of Pharmacopeias.

Claims

1. A hemostatic powder, comprising 18.6-20.2% COOH, 0.2-0.4% aldehyde, and 0.1-0.25% nitrogen containing regenerated oxidise cellulose powder impregnated with antifibrinolytic tranexamic acid and 6-aminocaproic acid.

2. The hemostatic powder according to claim 1, further comprising 0-2.2% Ca.sup.+2 and 0-1.1% Na.sup.+1 ion.

3. The hemostatic powder according to claim 2, further comprising 1.5-2.1% Ca+2 and 0.8-1.1% Na.sup.+1 ion.

4. The hemostatic powder according to claim 1 further comprising 0.1-0.8% antifibrinolytic tranexamic acid or 6-aminocaproic acid.

5. The hemostatic powder according to claim 1, further comprising antimicrobial active substance.

6. The hemostatic powder according to claim 5, wherein the antimicrobial active substance is selected from the group consisting of Rifampicin, Gatifloxacin, Doxycycline, Levofloxacin, Lincomycin, Clindamycin, and Ciprofloxacin.

7. The hemostatic powder according to claim 5, wherein the antimicrobial active substance is impregnated in a range of 2.3-2.9%.

8. The hemostatic powder according to claim 1, further comprising antimicrobial Bismuth complex.

9. The hemostatic powder according to claim 2, further comprising antimicrobial active substance.

10. The hemostatic powder according to claim 9 wherein the antimicrobial active substance is Rifampicin and doxycycline.

11. The hemostatic powder according to claim 10 wherein the doxycycline is impregnated in a range of 1.8-3.5%.

12. A hemostatic material, comprising regenerated oxidised cellulose cotton or cloth, containing COOH between 18.6-20.1%, at most 0.2% aldehyde, and at most 0.1% relative nitrogen, wherein the nitrogen has a loss on a drying rate of 2.3% and is impregnated with antifibrinolytic tranexamic acid and 6-aminocaproic acid with a hemostatic time of 7-12 s.

13. The haemostatic material according to claim 12, further comprising Ca.sup.+2 ions between 0-2.2% and Na.sup.+1 ion between 0-2.1%.

14. The haemostatic material according to claim 12, further comprising Ca.sup.+2 ion between 2.1-2.2% and Na.sup.+1 ion between 1.8-2.1%.

15. The haemostatic material according to claim 12, wherein the nitrogen is impregnated with antifibrinolytic tranexamic acid or 6-Aminocaproic acid at a concentration of 0.05-0.1%.

16. The haemostatic material according to claim 12, further comprising antimicrobial active substance.

17. The haemostatic material according to claim 12, wherein the antimicrobial active substance is selected from the group consisting of Chlorhexidine, Ciprofloxacin, Gatifloxacin, Levofloxacin, Rifampin, Lincomycin, Clindamycin, and Doxycycline.

18. The haemostatic material according to claim 12, wherein the antimicrobial active substance is impregnated at a concentration of 2.4-3.5%.

19. The haemostatic material according to claim 12, further comprising a Bismuth complex.

20. The haemostatic material according to claim 12, further comprising antibacterial active substance.

21. The haemostatic material according to claim 12, wherein the antimicrobial active substance is Rifampicin or Ciprofloxacin.

22. The haemostatic material according to claim 21, wherein Rifampicin or Doxycycline is impregnated at a concentration of 2.8-4.5%.

23. A method of producing a hemostatic powder, comprising the steps of putting 28-30DN viscon string woven or viscon fiber on a mixture of H.sub.3PO.sub.4 and HNO.sub.3 in two flasks; adding NaNO.sub.2 as powder into the mixture; keeping a reaction temperature at room temperature for 38-40 hours via aeration from an inlet of one of the two flasks; adding demineralised water 6 times of a volume of the flask and mixing; filtering with small pored gauze; rinsing with 200 ml water every time; keeping rinsing till pH of flushing water is 2.8-3.1; washing with 100 ml 98% ethanol and drying at room temperature; and drying at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 hours.

24. A method of producing a hemostatic powder, comprising the following steps: putting raw viscon woven on a mixture of H.sub.3PO.sub.4 and HNO.sub.3 in a range of 1/15 in a flask A of a glass reactor; adding NaNO.sub.2 into the mixture, wherein an acid concentration is 12-15/1000; passing gas obtained from the flask A to a flask B for a period of 120-170 hours, wherein the flask B contains material dried at 50 C. on P.sub.2O.sub.5 for 2 hours; heating the flask B with 0.5 l/h air on an outlet of gas; wherein when saturation of NO.sub.2 gas is reached, color of the material in the flask B becomes red-brown; providing the material with a gas ratio in the range of <2:07-2:1.05>; cleaning the gas by aspiration during closing of an inlet valve and opening of an upper valve (E); taking oxidise textile or woven viscon on separate plate and washing with demineralised water (with a ratio of 1:4) three times; when pH of flushing water is 2.8-3.2, adding 150 ml water and 4 ml 30% H.sub.2O.sub.2 for 40 g filtering after 1 hour; washing 150 ml water and 150 ml 98% alcohol once and drying at room temperature; and drying at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 hours.

25. A method for producing a gellation of a hemostatic powder, comprising putting 28-30DN viscon string woven or viscon fiber on a mixture of H.sub.3PO.sub.4 and HNO.sub.3 in two flasks; adding NaNO.sub.2 as powder into the mixture; keeping a reaction temperature at room temperature for 38-40 hours via aeration from an inlet of one of the two flask; adding demineralised water 6 times of a volume of the flask and mixing; filtering with small pored gauze; rinsing with 200 ml water every time; keeping rinsing till pH of flushing water is 2.8-3.1; washing with 100 ml 98% ethanol and drying at room temperature; and drying at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 hours; wherein 5-10% chitosan is used.

26. The method according to claim 25, wherein a ratio of chitosan to deacetyl is more than 85%.

27. The method according to claim 26, wherein gel is added into the water in a range of 10-50% and mixed at 300 rpm.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0091] FIG. 1. Gas phase oxidise cellulose production reaction system

[0092] A: NO.sub.2 gas production, 2 l reactor with coil

[0093] B: Fiber or woven material reactor, 5 l

[0094] C: (On the cooling 0-3 C. liquid circulates) B vessel cooled by circulating occur N.sub.2O.sub.3 gas, every 5 h, 2 l aeration, N.sub.2O.sub.4 circulate and recover in A vessel.

[0095] D: Tap is for pressure control

[0096] E: The tap of gas aspiration on the reactor

[0097] FIG. 2. With qualitative method, 5 different textile studied. Under anti-microbial control, obtained photos: (1) REOXC-2, (2) REOXC-1, (3) REOXC*CaNaT, (4) PA-RB, (6) Textile

[0098] c. S. aureus ATCC 6538 strain, 24 h incubation

[0099] d. E. coli ATCC 25922 strain, 24 h incubation

[0100] FIG. 3. Photograph of Hemostatic test example

DETAILED DESCRIPTION OF INVENTION

3. The Preparation of Formulation

[0101] In present invention 3 different formulation is prepared for hemostatic, antiseptic biocompatible samples preparation. [0102] Powder formulation (Pulver: samples coded PA) [0103] Woven or textile formulation (samples coded REOXC) [0104] Gel formulation (samples coded GA)

[0105] Every three formulation have the matrix material which has 18.6-20.2% COOH groups of regenerated oxidised cellulose (REOXC). Every three formulation plan to add 0-0.2% gelatine as 90-100% gelation, 0-3.2% Ca+2 ion as blood coagulation, 0-0.9% Na+1 as speeder for biodegradation and 0.1-1.1% antiplasmine, antifibrinolytic 6-aminohexanoic acid, 4-aminomethyl cyclohexanecarboxylic acid (trenexamic acid) or aprotinin. Easy biodegradation of oxide cellulose is carried certain rated Ca+2 and Na+1 ion. If Na ion is more than 1%, it causes low haemostat feature and haemostat time decreasing is determined. It is determined via in vivo test that using 0.1-1% trenexamic acid as antiplasmin is provided the haemostat stabilisation (Table 7-11). However Vladimir N. F. and Vlademir R, US 2008/0181936 recorded that 2,3-dialdehit cellulose carrier matrix obtained NaIO4 oxidation, this studies had used chlorhexidine, AgNO3 and lysozyme as anti-microbial; trenexamic acid, 6-aminocaproic acid as haemostat stabiliser. In present invention we have used first time Bismuth Oxidise cellulose compound as antimicrobial properties instead of Ag-oxidised cellulose, AgNO3, chlorhexadine. We tested to add siprofloksasin, gatifloxacin, levofloxacin, rifampin, lincomycin, clindamycin, doxisiklin as anti-microbial to have good synergy with Bismuth Oxidise and also it is tested at the first time. In this study siprofloksasin, gatifloxacin, rifampin and doxisiklin as antibiotic have been preferred. Hemostatic-antiseptic formulation is studied on preferably 1.2-2.4% bi-oxidise cellulose and 1-2.4% antibiotic.

[0106] On basic matrix of powder formulation it is predicted to use that REOXC which is haemostat and partly antiseptic and 1.2-1.8% Ca ion for speeder of the haemostat and 6-aminohexanoic said or 4-aminomethyl cyclohexanecarboxylic acid for decreasing the fibrin solubility. Samples prepared have 0.2-1.2% ratio. If the product will use for internal wound, 0.5-1.1% Na ion must be added. Woven and textile REOXC formed band impregnated calcium is done firstly by calcium acetate as mentioned on US2008/0181936 and sodium impregnation is done by sodium acetate. Standardisation problem of sodium rates is noticed therefore in present invention we have prepared sodium-2ethykhexanoat on organic phase and it is used for impregnation sodium quantitatively. After adding calcium and sodium and drying the band, antiplasmin is impregnated to textile. For antiplasmin preparation, 6-aminohexanoic said or 4-aminomethyl cyclohexanecarboxylic acid is solving in 40-55% isopropanol:water mixture. Specific antifibrinolitic compound have been determined by HPLC methods in accordance with European and US Pharmacopeia.

3.1. Oxidised Cellulose Product's Standardisation

[0107] NMR studies: On H1-NMR-C13-NMR studies show us the existence of COOH and aldehyde after polymer CDCl3 is solved in and alcohol and carboxyl group persililation with trimethylsily chloride/hexamethyldisilazane.

3.2. Carboxyl Quantitative Determination

[0108] It is recorded USP at 1995. 0.5 g sample (with P2O5 is dried in vacuum oven at 500 C for 1.5 h) is taken. Mix with 50 ml 2% calcium acetate for 30 min. Mixture titrated by using 0.1N NaOH (std) and phenolphthalein indicator. Amount of NaOH used in reaction is verified as blank. Using following formula carboxyl determine:

[0109] Carboxyl group (w/w %)=(NVMW.sub.COOH)/sample weight (mg)100

[0110] N: NaOH normality=0.1 N

[0111] V: used NaOH amount (ml) in the reaction

[0112] MW.sub.COOH=0.9

3.3. Aldehyde Quantitative Determination

[0113] 2 g oxidise cellulose sample (with P2O5 is dried in vacuum oven at 500 C for 1.5 h) get the reaction with 0.2 M 100 ml NaCl and adjust pH=2.9-3.01 with 0.1 M acetic acid. Place the sample on room temperature and dark for 72 h. Filter and wash with water to separate the acid. The wet sample is dried by air and place in dark room till the determination of carboxyl amount which is done mentioned at 3.2. Primary carboxyl value (at 3.2) subtract this carboxyl value and result is aldehyde value.

3.4. Molecule Weight Determination

[0114] SBP cellulose samples processed liquid ammonia or unprocessed, solve in 1 M cupriethylenediamine diluted from 3 M stock solution (Prolabo, France). Viscometric average polymerisation degree of cellulosic samples (DP.sub.V) is calculated by this formula (Rinaudo 1968);

[]=0.891DP.sub.v.sup.0.936

[0115] Molecular weight of cellouronic acid is determined by using HPSEC. Sodium salt (1 g/l concentration) of cellouronic acid is solved in water, and filter 0.22 um and injected to Shodex OH park SB 804 Hq and SB 805 HQ in Alliance GPCV 200 (Waters Tech., MA, USA) system. 0.2 M NaNO3 is used as diluter. And then the solution is analysed using 3 detectors (differential refractometer, multicapillary viscometer, multi angle laser light scattering DSP-F (Wyatt Tech. CA, USA) and specific index dn/dc=0.1512 at 25 C. Obtained oxidise cellulose polymer is 5103-1.5104 dalton determined.

3.5. Carbonyl Determination

[0116] 0.5 g oxidise cellulose sample (with P.sub.2O.sub.5 is dried in vacuum oven at 50 C. for 1.5 h) put in 250 ml flask. 50 ml hydroylxamine hydrochloride put in another 250 ml flask. One of flask must have drain tap and connect each other. Flasks are connected to water pump. Close tap and hydroylxamine hydrochloride solution pour REOXC's flask. Place the mixture at 50 C. for 2 h. Cool down the room temperature, take 25 ml and titrate to 0.1 N HCl pH 3.2. Another 25 ml solution titrate for blank. Calculate the carbonyl using this formula:

Carbonyl (% w/w)=MW.sub.co(BS)/10(Weight of sample)100 [0117] B and S is ml, and amount of 0.1 N HCl consumed Blank and Sample

[0118] For hydroxamine hydrochloride preparation, 50 g Hydroxamine is solved in HCl 120 ml 1N NaOH and add on 1000 ml water for dilution. Generally ketone carbonyl does not exist in REOXC is produced by NO.sub.2 oxidisation (CO<0.01).

3.6 Elementary Analysis

[0119] Carbon, Hydrogen, Nitrogen and Sulphur Elemental Determination:

[0120] They are done by Ankara University, Pharmaceutical Fac. Instrumental Analysis Lab. Analysis result especially for nitrogen is given.

[0121] Na. Ca, Zn, Bi, Ag Element Determination:

[0122] Using Atomic Absorption method they are done by Hacettepe Univ. Analitik Chemistry Dept.

3.7. Antifibrinolytic Treneksamikacid and 6-Aminocaproin Acit Determination

[0123] Qualitative analyses is used HPLC method which recorded on EU Pharmacopeia 5.0 (2005).

3.8. Cytotoxicity, Biosensitivity and Irritation Tests

[0124] Hemostatic, antiseptic REOXC products must be biocompatible therefore test cytotoxicity, biosensitivity and irritation values and must be in certain limits. WO 2010/086616, USP-XXI and EU Pharmacopoeia 5.0 (2005).

[0125] These test of products are done by Hacettepe University Pharmacy Fac. Pharmacology Dept.

[0126] Cytotoxicity:

[0127] Used standards are TS EN ISO 10993 medical device evaluation, TS EN ISO 10993-1 evaluation and trial, TS EN ISO 10993-5 in-vitro cytotoxicity assay, TS EN ISO 10993-12 Sample and reference material preparation.

[0128] Test method: (extract test) original extract contact with single layer cells 50-80% plating during 24 h.

[0129] Culture media: Eagle (non polsar)

[0130] Cell line: CCL 81 (Vero African Monkey Kidney Cell Line)

[0131] Extraction method: 0.2 g/ml test sample with media placed in incubator 5% CO.sub.2 (V/V) at 37 C. for 24 h. Negative control with nitrocellulose; positive control with natural rubber latex, reactive control with culture media w/o serum was done.

[0132] Evaluation criteria: After 24 h. incubation, costars observe under the microscope. Biological reactivity is evaluated in accordance with following table.

TABLE-US-00004 Grade Reaction Culture media status 0 none no separate cytoplasm granules, no cell growth decreasing, no cell lysis 1 very little seldom cell lysis, non circular cell more than 20%, weak bounding and no cytoplasm granule and no changing on morphologic, only little growth inhibition 2 some Circular cell count is less than 50%, no cytoplasm granule, observable cell inhibition is not more than 50% 3 mediocre Circular or cell lysis counts are not more than 70%, cell layers are not completely divided 4 harsh cell layers are completely or almost completely lysed.

[0133] Results numeric value: If the sample's grade is bigger than 2, it is cytotoxic; if smaller, it is noncytotoxic.

[0134] Sensitivity:

[0135] TS EN ISO 10993 medical device evaluation, TS EN ISO 10993-1 evaluation and trial, TS EN ISO 10993-5 in-vitro cytotoxicity assay, TS EN ISO 10993-10 Irritation and delayed type erethism tests.

[0136] Study: Negative control (2525 mm four layered textile)

[0137] Test procedure: 300-500 g, healthy, mature, same race female and male albino rats. Ten animals for test, five for control test is used. Back hairs are shaved.

[0138] Induction phase: Direct application of test and control samples on shaved area, wait for 6 h. and take off. These procedure applied 3 days per a week during 3 weeks.

[0139] Challenge phase: On 14. days, test samples all animals of test and control groups applied only test sample to the shaved area and not applied area and take off after 6 h.

[0140] Evaluation criteria: After taking off challenge patch, test areas at 24. and 48. hours is evaluated by Magnusson and Kligman scale (TS EN ISO 10993-10; irritation and delayed type erethism tests).

TABLE-US-00005 TABLE 7 Magnusson and Klisman Scale Test Reaction Graudate no change visible 0 spread-partly erethism 1 mild and nonspread erethism 2 intense erethism and/or inflation 3 * Subject group with non-erethism shows us no sensitivity

[0141] Skin Irritation:

[0142] Working design: 2525 mm four layer textile sample which added negative control, 0.9% saline solution.

[0143] Test procedure: 3 healthy adult rabbit, female and male 2-3 kg. One day before back of animals is shaved. Sufficient distance on two side of backbone as test area are selected for application and observation. If test sample is powder, add 0.5 ml 0.9% saline solution and apply two each on two sides. And also negative control is applied two each on two sides. Applied area cover with 2525 mm gauze and with semi-permeable bandage for 4 h. After contact time take off the bandage and mark the applied area.

[0144] Evaluation criteria: After taking off patch, at following 1., 24., 48. and 72. h is observed for erethism and oedema. Irritation grade is average test and negative control for each applied area. Irritation score is obtained by divide three observe grade at 24., 48., 72. to all irritation grade. Primer irritation index calculate to divide primer irritation score to animal count. Results are evaluate correspond to table below.

TABLE-US-00006 Scoring for Skin Reaction Reaction Irritation score Erethism and scar forms no erethism 0 dull erethism (visible hardly) 1 clear limit 2 mild erethism 3 scar form to prevent sharp erethism observation 4 Oedema formation no oedema 0 dull oedema (visible hardly) 1 clear limited oedema 2 mild oedema (approx. 1 mm) 3 sharp oedema (wider than 1 mm and application area) 4 Most sharper scar for irritation 5 [0145] All changes observed will be recorded.

[0146] Results:

[0147] During all test done cytotoxicity's result are +1; raw REOXC is +2 and after H.sub.2O.sub.2 stabilisation of product and after rinsing, result is +1 observed and is determined noncytotoxic. All samples are non-irritant observed. On skin irritation tests, primer irritation index of all samples are 0-0.28 determined, skin irritation is not observed.

3.9. Measurement of Hemostatic Properties

[0148] As we mentioned on introduction surgical operation and trauma can cause health risk, massive bleeding. After determine bleeding area, can stop with cauterisation and ligation and similar application, most important point is ongoing haemorrhage.

[0149] For surgical application, there are local effective haemostat products in the market. These are hydrogel, polyglucoronic (PAGA) and/or polyglucosamin derivatives. Coagulant materials, antimicrobial compounds, anti-inflamatation, analgesic and antihistaminic have been added. Gelatine, collagen spanch, oxidise cellulose, fibrin binding, natural biological polysaccharide are used frequently as absorbable haemostat (Horio and ark., 2001l.sup.it1, Murakami and ark., 2008.sup.lit2, Murakami and ark., 2009.sup.lit3).

[0150] Production development on this area must have biological compatibility, stop bleeding within the shortest time, preventing the exudation, not cause to stick inter tissue, speed up tissue healing and absorption features (World Intellectual Property Organisation, 2011.sup.lit4).

[0151] During the haemostat production development, rat carotid artery transection and liver laceration studies as in-vivo animal tests are done (World Intellectual Property Organisation, 2009, 2011.sup.Lit5,4).

Literatures

[0152] 6. Horio T, Ishihara M, Fujita M, Kishimoto S, kanatani Y, Ishizuka T, Nakamura S, Tanaka Y, Morimoto Y, Maehara T (2010). Effect of photocrosslinkable chitosan hydrogel and its sponges to stop bleeding in a rat liver injury model. Artificial Organs, Vol 34(4), 342-347. [0153] 7. Murakami Y, Yokoyama M, Nishida H, Tomizawa Y, Kurosawa H (2008). A simple hemostasis model fort he quantitative evaluation of hydrogel-based local hemostatic biomaterials on tissue surface. Colloids and Surface B: Biointerfaces, 65, 186-189. [0154] 8. Murakami Y, Yokoyama M, Nishida H, Tomizawa Y, Kurosawa H (2009). In Vivo and In Vitro evaluation of gelation and hemostatic properties of a novel tissue-adhesive hydrogel containing a cross-linkable polymeric micelle. Journal of Biomedical Material Research, Part B: Appl Biometer 91B, 102-108 (Wiley Periodicals) [0155] 9. World Intellectual Property Organisation (2011). Hemostatic agents and wound dressings. WO 2011/084326 A2 (International Publication Number) [0156] 10. World Intellectual Property Organisation (2009). Biocompatible and biodegradable biopolymer matrix. WO 2009/072146 A1 (International Publication Number)

3.10.1. Application of Haemostat Tests

[0157] Ethical Committee Approval:

[0158] Decision No:

[0159] Bakent niversitesi Tip ve Salik Bilimleri Aratirma Kurulu ve Hayvan Deneyleri Etik Kurulu'nun Jan. 4, 2013 tarih ve 13/24 sayili karari [0160] Place: Bakent niversitesi Deney Hayvanlari retim ve Aratirma Merkezi laboratuvarn. [0161] Starting date of all animal tests: 7 May 2013

[0162] All animal tests is acted upon instructions of Bakent niversitesi Deneysel Aratirma lkeleri.

[0163] Animals Material:

[0164] 64 ea. S. Dawley/W. Albino, 370-450 g male rats supplied by Bakent University, Laboratory Animals Growing and Research Centre. Rats were feeding with standard rat feed and fresh water.

[0165] Animal Classification:

[0166] There are two stages. At the first stage 32 prototype product tested on medial and lateral liver lobes laceration of 38 rats for haemostat properties. After tests, 4 prototype product which have appropriate coagulation product, 2 product in the market and 4 control animals are compared. The aim of this stage is determination of most effective powder and spanch prototype product to get the criteria of clotting time.

[0167] At the second stage, 4 prototype product which determine at the first stage is compressed on the cut of 12 rat medial liver lobe. Histopathologic evaluation was done indelibly at 5. and 12. days. For 7 prototype products which determined at first stage, clotting time measurement is repeated with 6 rats injected heparin. The aim of second stage test are recorded histopathological changes of visceral organ and determination of adhesion forms, biological incompatibility, resorption, morphological changes, internal bleeding after certain time.

[0168] Anaesthesia and Heparin Application Protocol:

[0169] All animals studied on, is anaesthetised with intraperitoneal xylazine hydrochloride (6 mg/kg) and ketamine hydrochloride (60 mg/kg). After anaesthesia, for 6 animals, lateral tail vein catheterisation is done and injected 500 IU/kg heparin. After 15 min, laparotomy operation is started.

3.9.2. Surgical Operation

[0170] First Test Stage:

[0171] Anaesthetised rats are fixed on cork board. Ventral abdominal section are shave and disinfected. Under the aseptic conditions, made an incision 3-4 cm. mid line on thorax xiphoid approx. 2 cm near caudal through the abdominal cavity. Liver lobs is taken out incision line. After filtering the liquid, put the stretch film between liver lobs and visceral and for determination the bleeding, put the filter paper between the lobs. For every rat median and lateral 1 cm length, 0.3 cm depth incision is done separately. 30 s. blood flow for 45 degree angled rats are observed and compressed by prototype products and equivalent product for 5 min. excluding negative control groups. During 5 min. bleeding and blood on filter paper is recorded. FIG. 3

[0172] Second Test Stage:

[0173] 4 prototype product determined at first stage and the animals injected heparin are tested specified in first stage. Operation incision stitched and kept animals alive. After operation at 5. day after one each group and at 12. day others was done euthanasia and took the samples of necropsy, histopatholgical. [0174] Data statistical analysis is evaluated by student-t tests.

TABLE-US-00007 TABLE 4 Haemostat values of 14 textile spanch prototype product and 2 equivalent product is tested. Applied amount Hemorrhage Bleeding time Code Test number (cm cm) (gr) (s) COOH % REOXC-(1) 6 2 4 0.418 10-15 19.80 REOXC-CaNa 2 2 4 0.230 10-20 19.20 REOXC-(2) 6 2 4 0.019 10-20 19.35 REOXC-CaNaT 3 2 4 0.420 10-30 19.25 PA-RB 2 2 4 0.414 within 120 19.58 REOXC-CaT(1) 1 2 4 0.659 30 20.00 REOXC- 2 2 4 0.418 in the range of 18.50 CaNaT/Zn 90-180 REOXC-CaT(2) 2 2 4 0.332 in the range of 20.00 40-80 REOXC- 2 2 4 0.627 <80 19.35 CaNaT/Bi REOXC-CaNa 1 2 4 0.309 80 18.50 REOXC-CaT(3) 6 2 4 0.418 10-12 19.35 PA-SB 3 2 4 0.432 90 19.58 Equivalent 2 2 4 0.272 90-150 17.50 product 1 (US) Equivalent 2 2 4 not work ineffictive 15.20 product 2 (TR) n = 24 rats, 2 prototype product test for each Hemorrhage = (Weight of filter paper blood absorbed) (Nature filter paper weight)

TABLE-US-00008 TABLE 5 Hemostatic values of 8 ea. prototype powder product Test Applied Hemorrhage Bleeding Cod number amount (g) (g) time (s) Description PA-1 2 0.210 0.911 10-20 PA-2 3 0.210 ineffective PA-3 2 0.210 0.280-0.658 55 PA-4 8 0.210 0.185-0.979 10-45 PA-5 7 0.210 0.153-0.987 10-35 PA-6 1 0.210 0.268 25 PA-7 3 0.210 40-70 PA-8 1 0.210 25 PA-9 2 0.210 6-10 HG-1 2 0.210 45-65 HG-2 ineffective HG-3 55-75 HG-4 25-45 HG-5 25-35 HG-6 45 HG-7 45-75 HG-8 25-35 HG-9 13-20 n = 14 rats, 2 prototype product test for each Hemorrhage = (Weight of filter paper blood absorbed) (Nature filter paper weight)

TABLE-US-00009 TABLE 6 As a result go first stage tests, hemostatic values after I.V. heparin injection. applied amount Bleeding Code Test count (g or cm cm) time (s) Description PA-4 1 0.400 g 110 PA-5 1 0.200 g 115 REOXC S 1 2 4 38 REOXC-Ca 1 2 4 33 Spanch REOXC-CaT S 1 2 4 40 PA-9 1 0.200 g 15 n = 6 rats

TABLE-US-00010 TABLE 7 Haemostatic values of 4 prototype products is applied on liver lob incision. Code no. (n) applied amount Bleeding time(s) PA-4 powder n = 3 0.210 g 10-20 s PA-5 powder n = 3 0.210 g 10 s REOXC S n = 3 2 4 cm 10-15 s REOXIC-CaT S 2 4 cm 10-15 s *S = spanch

TABLE-US-00011 TABLE 8 Chosen4 prototype after first stage tests applied liver lob incision, necropsy and histopathologic value of following 7. day of alive animals: Code no (n) Necropsy indications Histopathologic indications PA-4 powder All animals in this group: There are connective tissue increasing on (n = 1) alive glisson capsule and coagulation necrosis at internal bleeding negative the centre, giant cell, lymphocyte, powder applied absorbed macrophage around. Outmost tissue is completely unnatural granulation. Degenerative changes incision point has whitened on the centre of lob is make attention. tissue appearance Neutrophile leucocyte is observed on the portal region. All vein and sinusoid is filled with blood. PA-5 powder All animals in this group: There are fibrosis and thickening of (n = 1) alive unnatural granulation tissue. Together with internal bleeding negative phagocytosed macrophage which get blue applied material absorbed colour. Hepatocyte degenerative changes completely and mononuclear cells are observed. All vein incision point has whitened and sinusoid is filled with blood. tissue appearance. But less than PA-4. REOXC *S All animals in this group: Thickening of unnatural granulation tissue (n = 1) alive and little mono nuclear cell infiltration, internal bleeding negative fibrosis on glassine capsule are observed. applied material absorbed Hepatocyte degenerative necrotic changes completely and bleeding is noticed. Mono nuclear cells on portal region is observed. All vein and sinusoid is filled with blood. REOXC-CaT All animals in this group: Fibrosis on glisson capsule, little mono *S (n = 1) alive nuclear cell infiltration, material phagocytose internal bleeding negative macrophage powder applied absorbed and thickening of unnatural granulation completely tissue is observed. At same time fibrin strings, bleeding and neutrophile leucocyte is available. Hepatocyte degenerative and necrotic changes, pencil and increment on kupffer cell. All vein and sinusoid is filled with blood. *spanch

TABLE-US-00012 TABLE 9 Chosen 4 prototype after first stage tests applied liver lob incision, necropsy and histopathologic value of following 15. day of alive animals: Code no (n) Necropsy indications Histopathologic indications PA-4 All animals in this group is alive Fibrosis on glisson capsule, veining, powder Feeding is good little mono nuclear cell infiltration and (n = 2) Internal bleeding is negative thickening of unnatural granulation Applied material is absorbed completely tissue and material phagocytose Incision is clean and healing macrophage is observed. Hepatocyte Determine no conglutination of liver degenerative changes are noticed. tissue and other abdominal organs Mono nuclear cells on portal region is Conglutination of omentum tissue to observed. All veins and sinusoids are wound is partly determined. filled with blood. Any pathologic case and liquid on other visceral abdominal organ is not available. PA-5 All animals in this group is alive Fibrosis on glisson capsule, veining, powder Feeding is good little mono nuclear cell infiltration and (n = 2) Internal bleeding is negative thickening of unnatural granulation Applied material is absorbed completely tissue and material phagocytose Incision is clean and healing macrophage is observed. Hepatocyte Determine no conglutination of liver degenerative changes are noticed. tissue and other abdominal organs Mono nuclear cells on portal region is Conglutination of omentum tissue to observed. All veins and sinusoids are wound is partly determined. filled with blood. Any pathologic case and liquid on other visceral abdominal organ is not available. REOXC *S All animals in this group is alive Fibrosis on glisson capsule, veining, (n = 2) Feeding is good little mono nuclear cell infiltration and Internal bleeding is negative thickening of unnatural granulation Applied material is absorbed completely tissue and material phagocytose Incision is clean and healing macrophage is observed. Hepatocyte Determine no conglutination of liver degenerative changes, bleeding are tissue and other abdominal organs noticed. Material phagocytose Conglutination of omentum tissue to microphage, mono nuclear cells on Glisson capsule and liver medial lob is adipose tissue and neutrophile partly determined. leucocyte on portal region is Any pathologic case and liquid on other determined. All veins and sinusoids visceral abdominal organ is not are filled with blood. available. REOXC-CaT All animals in this group is alive Fibrosis on Glisson capsule, mono *S (n = 2) Feeding is good nuclear cell infiltration, material Internal bleeding is negative phagocytose macrophages and Applied material is absorbed completely thickening of unnatural granulation Incision is clean and healing tissue formation is observed. Determine no conglutination of liver Hepatocyte degenerative changes and tissue and other abdominal organs mono nuclear cells on sinusoids is Conglutination of omentum tissue to noticed. All veins and sinusoids are Glisson capsule and other abdominal filled with blood. organs is not available. Available for other samples is not observed. But conglutination of medial lobe to incision is lightly determined. Any pathologic case and liquid on other visceral abdominal organ is not available.

[0175] Result:

[0176] 4 prototype products (PA-4, PA-5, REOXC S, REOXC-CaT S) choose on preliminary test, are effective haemostat as a result of animal test and applications. Although all materials have acceptable limits for inflammation, fibrosis and necrosis, REOXC-CaT S and PA-5 powder products have better results for tissue reaction.

3.10. Antimicrobial Tests

[0177] Bacteria Culture Preparation:

[0178] On the study, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis DSM 20044, Escherichia coli ATCC 25922, Acinetobacter baumannii DSM 30007 and Pseudomonas aeruginosa ATCC 27853 is used. ATCC strain is supplied by American Type of Culture Collection (Wesel, Germany), DSM strainis supplied by Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

[0179] Bacteria lyophilised, is diluted according the instruction of manufacturer. Sheep blood agar is incubated at 37 C. for 24 h. (Salubris A.S. Turkey). Two passages are done. After than Brain Heart Infusion (BHI) liquid medium (BBL, BD Diagnostics, USA) 0.5 McFarland turbidity is adjusted 10.sup.8 cfu (colony units/ml) and serial dilution inoculum amount is adjusted 10.sup.6 cfu/ml.

[0180] Determination of Minimal Inhibitor Concentration (MIK) of Powder Products:

[0181] 200 mg of every product is diluted in 10 ml BHI liquid medium (20 mg/ml). 5 tubes contained 4.5 ml BHI liquid medium is prepared. One of them put 0.5 ml diluted product and serial dilution is done. 5 dilution tests between 0.2 mg/ml and 210.sup.4 mg/ml concentration is studied. 0.5 ml 106 mg/ml bacteria solution is added. Last bacteria concentration in tube is 10.sup.5 cfu/ml. After incubation at 37 C., for 24 h, no growth is defined as MIK.

[0182] Determination of Powder Products Antimicrobial Activities:

[0183] 200 mg of every product is diluted in 10 ml BHI liquid medium (20 mg/ml). 5 tubes contained 4.5 ml BHI liquid medium is prepared. One of them put 0.5 ml diluted product and serial dilution is done. 0.5 ml 10.sup.6 mg/ml bacteria suspension for every tube is added. After incubation at 0. h and 24. h, take 0.1 ml (10.sup.4 cfu/ml) from every tube, add to Nutrient Agar (Salubris A.S. Turkey). After incubation at 37 C. for 24 h, colony count. Positive control tube has no product but have 10.sup.4 cfu/ml bacteria, also incubated at the same condition and colony count. Antibacterial results are listed on Table-10.

TABLE-US-00013 TABLE 10 MIK* results of ten different products S. aureus S. epidermidis E. coli ATCC P. aeruginosa A. baumannii ATCC 29213 DSM 20044 25922 ATCC 27853 DSM 30007 (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) PA-1 >2 >2 >2 2 2 PA-2 >2 >2 >2 >2 >2 PA-3 >2 >2 >2 >2 >2 PA-4 >2 >2 >2 >2 >2 PA-5 >2 >2 >2 >2 >2 PA-6 >2 >2 >2 >2 >2 PA-7 <2 10.sup.3 2 10.sup.2 <2 10.sup.4 <2 10.sup.3 2 10.sup.2 PA-8 <2 10.sup.4 <2 10.sup.4 2 10.sup.1 2 10.sup.1 2 PA-9 2 10.sup.1 2 10.sup.1 2 10.sup.1 2 10.sup.3 >2 PA-10 >2 >2 >2 >2 >2 *Minimal inhibitor concentration (MIK) is the concentration of no bacteria, no colony count. This table show us PA-1 partly, but PA-7, PA-8, PA-9 is highly antibacterial.

[0184] Antimicrobial Activity Determination of Textile Material:

[0185] Bacteria Medium Preparation:

[0186] In the study for quantitative method, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 medium is used. Medium is tested for vitality and purity control, to passage blood agar.

[0187] Qualitative Method:

[0188] For this method, AATCC 147 standards is used. Test bacteria, 5 different textile and antibacterial effect of unprocessed textile as control by diffusion method is studied.

[0189] Every strain Mac Farland 10.sup.8 cfu/ml is prepared by the explained method above. After that spread plate technique is applied on Nutrient Agar (Salubris A.S. Turkey) by sterile swab and narrow lines. After spreading, put 5 textiles (22 cm, sterilised by ETO) and unprocessed textile as negative control at middle of plates. All plates incubated at 37 C. The plates is evaluated from the point of inhibition zone and bacteria growth. If there is inhibition zone, measure the zone as mm (Vytrasova et al. J Ind Microbial Biotechnol 2008; 35: 1247, Pinho E et al. Ann Microbiol 61:493-498).

TABLE-US-00014 TABLE 11 Five different products results after 24 h incubation in S. aureus ATCC 6538 and E. coli ATCC 25922 strains. S. aureus ATCC 6538 E. coli ATCC 25922 Products Zone (mm) Zone (mm) PA-RB 53 31 PA-SB 27 28 REOXC-CaNaT 31 24 REOXC (1) 27 NZ REOXC (2) 31 22 NZ: No inhibition zone These values show us zone (mm) has no growth. Measurement is done at the middle of edge of square inhibition zone which is composed peripheral of square textile. This table show us, PA-RB is most efficient against S. aureus and E. coli. One can see effective zones at FIG.-3.

4. Experimental Procedures and Samples

4.1 Basic Matrix Samples

Example-1. Regenerated Oxidise Cellulose Powder (Liquid Phase Oxidisation)

[0190] REOXC Pudr: REOXC-P (P=pudr)

[0191] Put the mixture of 50 ml H.sub.3PO.sub.4 85% and 75 ml HNO.sub.3 65%, 8 g viscon fiber or 55 cm 28DN woven viscon in the two necked flask. To keep temperature of reaction at 38-40 C., aerate from one neck of flask. Put cold demineralised water 6 times of flask volume and mix strongly. Filter with small pored gauze. Rinse with 200 ml water 3-5 times. Keep rinsing till pH of flushing water is 2.8-3.1. After then rinse with 98% ethanol and dry at room temperature. Then dry with P.sub.2O.sub.5 in vacuum oven at 50 C. for 2 h. Drying loss is 4-5%, COOH yield is between 20.0-21.8%; aldehyde yield is 0.3-0.5%, bound nitrogen is 0.1-0.25%. If change the conditions of rinsing (30% H.sub.2O.sub.2 and 98% ethanol), COOH yield is 21-22.2%, nitrogen is <0.05%, aldehyde yield is <0.1%.

Example-2. Regenerated Oxidise Cellulose Cotton and Textile (Gas Phase Oxidation) REOXC

[0192] Glass reactor at FIG. 1, Put 1000 ml 85% H.sub.3PO.sub.4 and 1500 ml 65% HNO.sub.3 on 5 l flask. Add 120 g of 150 g/m.sup.2 raw woven viscon to this mixture. 33 g NaNO.sub.2 is added slowly within 45 min. 400 g viscon cotton dried with P.sub.2O.sub.5, at 50-60 C. for 2 h (30DN woven, 30DN textile or 28 DN woven and 28 DN textile) is put 20 1 N.sub.2O.sub.4 gas obtained outlet of Flask A pass through to Flask B for 150-170 h. Flask A is heated at 45-50 C. for 1.5-2.5 h. Flask B is heated from gas outlet by 0.5 I/h air. Whenever colour of oxidation material on flask B is red-brown, it means N.sub.2O.sub.4 saturated. Cellulose material:gas rates must be (<2:0.6-2:1).

[0193] At the end of the reaction 2 no. tap on Flask B close and aerate from 3 no. tap and take off system. Gas is aspirated. Evolved gas is hold with wet CaO granule holder or by passing through to water. Put the oxidised textile or woven material on 10 l flask. Rinse with demineralised water (1:4) three times. Measure the rinse water. If pH 2.8-3.2, add on 150 ml water, 4 ml 30% H.sub.2O.sub.2 and wait for 1 h and filtrate. Rinse with 1.5 l water and once 1.5 l 98% alcohol and dry at room temperature. Put in vacuum oven with P.sub.2O.sub.5 at 50 C. for 2.5 h.

TABLE-US-00015 Product ready to pack: Dry loss 2.4% COOH 19.32-20.1% Aldehyde 0.2% N 0.11%

[0194] After aspiration, amount of nitrogen is 0.6-1.1%, but for final product it is 0.05-0.1% after rinsing. Before H.sub.2O.sub.2 reaction, aldehyde yield is 2.2-3.1% but this yield is almost 0.2-0.4% for final product. However Brisk T. S., Beverely H., Remanick A. H., Pasadena C. DP2061796 (1970) is recorded that the stabilisation of REOXC is done by NaBH.sub.4; we prefer Hydrogen peroxide stabilisation. This is advantage reflection for hemostatic feature (See Table. 2). And also COOH yield is increasing in the range of 0.2-0.5%. In general COOH value of the final product is 18.75-20.15%. This value is sufficient hemostatic feature for qualified textile according to USP. It is recommended that the temperature of B flask keep <19-26 C. during the reaction. N.sub.2O.sub.3 gas obtained after oxidation, at this temperature, is going to cooler with keeping gas phase. With 0.5 I/h air oxygen, it re-convert into N.sub.2O.sub.4 and feature in oxidisation.

[0195] On this invention we design the reactor (FIG. 2) for industrial production. COOH oxidisation is controlled kinetically by using certain amount of air oxygen. We determined during the validation studies that 70-100 g/m.sup.2 textile woven (DN=32, 30, 28), DN number of viscon cotton has important role; as a result COOH yield of REOXC for DN=28 is 18.6%, for DN=30 is 19.35%, for DN=32 is 20.30%. Preferably DN=30-32 viscon woven textile is predicted to use. If DN=28 will use, woven's density must be 75-80 g/m.sup.2. There is proportional rates between reaction time in the range of 130-168 h and COOH amount in the range of 17.9-20.15%. Process time is adjusted with COOH amount determination in every 24 h. Production of NO.sub.2 gas in 5 l reactor and 45-55 C. process temperature must be controlled. Till the colour becoming deep brown, this heated process keep on going and stop to heat. whenever the colour become light brown, heating must be started at 45-55 C. This is the kinetically control production.

Example-3. Adding Calcium and Sodium to Powder Oxidise Cellulose PA-3

[0196] a. 16.0 g REOXC pudr containing 21.1% COOH is solved in solution of 3.6 g NaOH in 175 ml demineralised water. Put 3.51 g CaCl.sub.2.2H.sub.2O compound with 25 ml demineralised water in solution of first step. After adding 4 ml 30% H.sub.2O.sub.2, put mechanical stirrer for 60 min. pH is adjusted with acetic acid for 5.3-5.4. After adding 175 ml. 98% ethanol, suspension is mixed for 20 min. For separation centrifuge at 4000 rpm for 8-10 min. Rinse with 60% ethanol three times and centrifuge. Take the sediment and put petri dishes and freeze at 40 C. and lyophilise for 24 h.

TABLE-US-00016 Product: COOH 21.15% Ca 2.0-2.1% Na 1.6-1.9% N 0.11% Aldehyde 0.2-0.3% [0197] b. If it is preferred that Na rates is smaller than 1.8%, pH will be adjusted 4.3-4.5 and obtain that Na yield is 0.9-1.1% and so hemostatic features increasing. At the same time bioabsorbability is increasing and optimising in good conditions. See hemostatic studies PA-3

TABLE-US-00017 Product: COOH 21.15% Ca 2.0-2.1% Na 0.9-1.1% N 0.11% Aldehyde 0.2-0.25%

Example-4. Preparation of Na-2-Ethyl Hexanoat

[0198] 14.6 g 2-Ethyl hexanoat is solved in 100 ml methanol. 4.0 g NaOH is solved in 100 ml methanol. These two solution is mixed and fill 500 ml up methanol which must be fresh distilled on KOH. And then Na-2-ethyl hexanoat solution in Methanol is prepared.

Example-5. Calcium and Gelatine Impregnated Powder Oxidise Cellulose PA-CG

[0199] Powder or textile oxidise cellulose which contain 15 g 20.1% COOH, is mixed with solution of CaCl.sub.2.2H.sub.2O in 1250 ml demineralised water, and is added 12 g 20% Na.sub.2CO.sub.3 and centrifuge 200 rpm for 30 min at 95 C. The mixture is cooled down 60 C. and is mixed with 4 ml 30% H.sub.2O.sub.2 at room temperature, adjust pH 5.3 with 20% acetic acid. 3 g gelatine is solved in 25 ml water and stand over night to provide the gelling and is added to main solution which must be 50 C. and is mixed strongly and slowly (300-500 rpm). Residue is cooled down 30 C. and add 150 ml 98% alcohol and separation is done by centrifuge 4000 rpm for 15 min. Rinse with 60% ethanol three times and take in petri dishes and is frost at 40 C., is lyophilised 18 h and then is dried at 50 C. on P.sub.2O.sub.5 in vacuum oven.

TABLE-US-00018 Dry loss 3.2% COOH 19.6-20.05% Ca 2.0% N 2.8% Na 1.2% Aldehyde 0.14%

4.2. Hemostatic and Antiseptic Samples

[0200] On WO2000/04939 A1 studies is determined that Zn.sup.+2, Ag.sup.+1, Bi.sup.+3, Na.sup.+1 ions are impregnated oxidise cellulose (linter cotton) contain 16.2% COOH; these compound can be used for gastrointestinal antiseptic. On our studies gelatine and chitosan is also used. Same process on WO2010/086616A1 studies is investigated that especially helicobacter pylori effect of product's anti microbial spectrum.

[0201] On present invention it is tested separately impregnated all five ions (Ca.sup.+2, Zn.sup.+2, Ag.sup.+1, Bi.sup.+3, Na.sup.+1) and they are standardised. It is important invention determined that the sampled called HA-9 (REOXC-Bi) impregnated with Zn.sup.+2, Ag.sup.+1, is quite good hemostatic, antiseptic properties with chosen antibiotics and additionally have synergic effect altogether.

Example-6: Oxidise Cellulose-Bi Compound REOXC-Bi HA-9

[0202] On this invention different techniques are used than WO 00/04939A1 which is applied for amine-dithiocarbamates.

[0203] The mixture of 9.52 g oxidise cellulose (contain 19.35-20.05% COOH) is solved in 1.9 g NaOH by mixing (Solution I). 9.7 g Bi(NO.sub.3).sub.3.5H.sub.2O compound is solved in 20 ml 2% HNO.sub.3 and 80 ml water (solution II). Firstly mix Solution I strongly and add in Solution II drop by drop within 3.5 h. This mixture centrifuge over night at 250-300 rpm. Add on 200 ml 98% ethanol and put +4 C. for 2 h. The dispersion is separated to centrifuge 4000 rpm for 10 min. Rinse with 80% ethanol two times and with 75% acetone and with 98% ethanol. Residue separated by centrifuge, is taken petri dishes and frost at 40 C. and is lyophilised for 12 h and then is dried at 50 C. in vacuum oven on P.sub.2O.sub.5.

TABLE-US-00019 Particle size 2-5 um Dry loss 2.4% COOH 19.65% Bi 16.5% N 3.5% pH 3.35 (1% suspension)

Example-7: Oxidise Cellulose-Zn Compound REOXC-Zn/Na

[0204] Solution of 1.8 g NaOH in 60 ml water is added oxidise cellulose which contains 10 g 19.35-20.05% COOH. 3.31 g ZnCl.sub.2 is solved in 20 ml demineralised water and add the solution drop by drop within 45 min. and mix at 250 rpm. Add 3 ml 30% H.sub.2O.sub.2 and mix 30 min at 300 rpm. pH 5.5 is adjusted with 20% acetic acid. 80 ml 98% ethanol is added and mix 10 min at 300 rpm and then centrifuge 10 min. at 4000 rpm. Rinse with 70% alcohol two times and disperse sediment is separated and frost 40 C. and lyophilised for 18 h and then is dried at 50 C. in vacuum oven on P.sub.2O.sub.2.

TABLE-US-00020 Particle size 3-5 um Dry loss 3.8% COOH 19.6% N 0.05% Na 2.5% Zn 8.2%

Example-8: Oxidise Cellulose-Ag Compound REOXC-Ag PA-10

[0205] Method determined WO2010/086616A1 is modified. [0206] a. Solution of 1.8 g NaOH in 60 ml water is added oxidise cellulose which contains 10 g 19.35-20.05% COOH. Add 3 ml 30% H.sub.2O.sub.2 and mix 30 min at 250 rpm. 8 g AgNO.sub.3 is solved in water. Add slowly to oxidise cellulose solution within 30 min. and mix at 300 rpm for 2 h. Residue is separated by centrifuge. Rinse with 2 ml 30% H.sub.2O.sub.2 in 50 ml water. Rinse with 80% ethanol two times and with isopropanol once. Dry at room temperature at first step and then dry at 50 C. in vacuum oven.

TABLE-US-00021 Particle size 5-8 um Dry loss 3.7% COOH 19.7% N 0.6% Na 0.4% Ag 18.2% pH 4.7 (1% suspension) [0207] b. Solution of 1.8 g NaOH in 60 ml water is added oxidise cellulose which contains 10 g 19.35-20.05% COOH. Add 4 ml 30% H.sub.2O.sub.2 and mix 30 min. 6 g AgNO.sub.3 is solved in 20 ml water. Add slowly to oxidise cellulose-Na solution and mix for 4 h. Residue coloured light brown is separated by centrifuge at 4000 rpm. Rinse with 2 ml 30% H.sub.2O.sub.2 in 50 ml water. Rinse with 80% ethanol two times and with 100 ml isopropanol once. Dry at room temperature at first step and then dry at 50 C. in vacuum oven for 3 h.

TABLE-US-00022 Particle size 4-7 um Dry loss 2.7% COOH 19.7%% N 8% Na 0.9% Ag 16.2% pH 15.7% (1% suspension)

4.3 Hemostatic Textiles

Example-9. Ca+.SUP.2 .Impregnated to REOXC REOXC-Ca

[0208] 200 ml demineralised water mix with 200 ml isopropanol and 1.171 g Ca(CH.sub.3COO).sub.2.2H.sub.2O is solved in this resolvent. Add 10 g REOXC woven which contains 19.6-20.01% COOH and then is evaporated at 25 C. for 1 h. Rinse with 50 ml isopropanol twice and dry at room temperature. Dry in vacuum oven at 50 C. on P.sub.2O.sub.5 for 2 h.

TABLE-US-00023 Dry loss 2.5% COOH 19.7-20.01% Ca 2.2-2.5% N 0.10%

Example-10. Ca.SUP.+2 .and Na.SUP.+1 .Impregnated to REOXC REOXC-Ca, Na

[0209] c. 170 ml demineralised water is mixed with 170 ml isopropanol and 0.984 g Ca(CH.sub.3COO).sub.2.2H.sub.2O and 1.32 g Na-acetat trihydrate is solved in this resolvent. Add 8.4 g REOXC which contains 19.5-20.05% COOH and then is evaporated at 25 C. for 1.5 h. Rinse with 50 ml isopropanol twice. Dry at room temperature and at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 h.

TABLE-US-00024 Dry loss 2.9% COOH 19.6-20.0% Ca 2.1-2.2% Na 1.8-2.1% N 0.12% [0210] d. 10.0 g REOXC-Ca (sample-9) is weighted and add the mixture of 20 ml demineralised water and 80 ml isopropanol. Add on 9 ml of sodium-2-ethylhexanoat in 16.8% methanol and 15 ml isopropanol. Mix them on evaporator for 10 min. Obtained product is rinsed with 50 ml isopropanol twice. Dry at room temperature and at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 h.

TABLE-US-00025 Dry loss 3.1% COOH 19.8-20.0% Ca 2.1-2.2% Na 1.4-1.5% N 0.11%

[0211] Note: Effective hemostatic, biocompatible and bio-absorbable product must have less than 1.1% sodium yield. As quantitative sodium impregnation is proper method, it is found that 4-6 ml 16.8% Na-2-ethylhexanoat solution is correct for 10 g REOXC.

Example-11. Impregnation of Anti-Plasmin 6-Aminocaproic Acid or Tranexamic Acid to REOXC-Ca,A, and REOXC-Ca.T

[0212] Take 10 g REOXC-Ca (Sample-9) and add on 0.5 g tranexamic acid or 6-aminocaproic acid is solved in 20 ml demineralised water and then evaporate with 30 ml isopropanol for 15 min. Stand at room temperature for 5 min. Obtained product is rinsed with 75 ml isopropanol twice. Dry at room temperature over night and then at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 h.

TABLE-US-00026 b. REOXC-Ca,T (T = Tranexamic acid) Dry loss 2.4% COOH 19.6% Ca 2.10% Tranexamic acid 0.8% N 0.91% b. REOXC-Ca, A (A = 6-aminocaproic acid) Dry loss 2.5% COOH 19.6% Ca 2.11% 6-aminocaproic acid 0.7% N 1.01%

Example-12. Impregnation of Calcium, Sodium, Tranexamic Acid and Zinc to Woven Oxicel REOXC-Ca,Na,T/Zn

[0213] 0.5 g Ca(CH.sub.3COO).sub.2.2H.sub.2O and 0.5 g Zn(CH.sub.3COO).sub.2.2H.sub.2O is solved in mixture of 170 ml demineralised water and 170 ml isopropanol. Add on 10 g oxicel contain 20% COOH and percolate for 2 h at room temperature. Rinse with isopropanol and Dry at room temperature. Obtained product is add on 50 ml ispropoanol and 5 ml 16.8% sodium-2-ethylhexanoat for 30 min. Take the product and rinse with isopropanol. Dry at room temperature. 0.5 g tranexamic acid is impregnated with as sample-11 techniques. Dry at room temperature and at 50 C. on P.sub.2O.sub.5 in vacuum oven for 2 h.

TABLE-US-00027 Dry loss 2.5% Ca 1.95% Na 1.4% Zn 2.05% N 0.82% Tranexamic acid 0.72%

4.4. Preparation of Powder Samples

Example-13. Regenerated Oxidise Cellulose (REOXC-P) PA-4

[0214] Take Example-1 as 20 g liquid phase oxicel and is solved in 3.75 g NaOH (in 100 ml demineralised water). Add on 4 ml 30% H.sub.2O.sub.2 and mix 30 min and mix for 30 min. pH=2.95-3.1 is adjusted with 20% acetic acid. Sediment product centrifuge at 4000 rpm. Rinse with 50 ml demineralised water and 50 ml 75% ethanol. Wet pat is put petri dishes. Keep at room temperature for 5 h. Frost at 40 C. and lyophilise for 18 h. Dry at 50 C. on P.sub.2O.sub.5 for 1.5 h in vacuum oven.

TABLE-US-00028 Particle size 3-7 um Dry loss 2.8% COOH 20.05% Na 0.14% N 0.1% Aldehyde 0.11%

Example-14. Powder Sample Contain Sodium, Calcium, Tranexamic Acid and Gelatine (REOXC-Na,Ca,T,P) PA-1

[0215] 10.0 g PA-4 mix with 10.0 g REOXC-Ca, Na and 0.35 g Tranexamic acid, 0.65 g gelatine, 120 ml 50% ethanol for 2.5 h. Add on 100 ml isopropanol and mix for 1 h. Centrifuge at 4000 rpm for separation. Wet product put petri dishes. Keep at room temperature for 3 h. Frost 40 C. and lyophilise for 18 h. Obtained product is dried at 50 C. on P.sub.2O.sub.5 in vacuum oven for 1.5 h.

TABLE-US-00029 Particle size 4-7 um Dry Loss 2.2% COOH 19.61% Ca 1.7% Na 1.2% Tranexamic acid 1.4% Gelatin 0.32% N 0.11% Aldehyde 0.11%

Example-15. Powder Sample with Sodium, Calcium, Tranexamic Acid, Gelatine and Zinc (REOXC-Na, Ca, R, T, G/Zn) PA-2

[0216] 10.0 g HA-4 mix with REOXC-Ca, Na and 0.35 g Tranexamic acid, 0.65 g gelatine, 0.5 g REOXC-Zn, 120 ml 50% ethanol for 2.5 h. Add on 100 ml isopropanol and mix at 3000 rpm for 1.5 h. For separation, centrifuge at 4000 rpm. Obtained product put petri dishes and keep at room temperature for 3 h. Frost at 40 C. and lyophilise for 14 h. Dry at 50 C. on P.sub.2O.sub.5 in vacuum oven for 1.5 h.

TABLE-US-00030 Particle size 3-6 um Dry loss 2.1% COOH 19.56% Ca 1.6% Na 1.1% Tranexamic acid 1.5% Gelatine 0.32% Zn 0.25% N 0.11% Aldehyde 0.11%

Example-16. Powder Product with Sodium, Calcium, Tranexamic Acid, Bismuth and Gelatin (REOXC-Na,Ca,T,G,Bi) PA-5

[0217] 12.0 g REOXC-Ca,Na mix with 0.42 g Tranexamic acid, 0.75 g gelatine, 1.2 g REOXC-Bi (PA-9), 120 ml 50% ethanol for 3 h (mechanically 250 rpm). Add on 100 ml isopropanol and mix for 1.5 h. Centrifuge at 4000 rpm. Gel product put petri dishes. Keep at room temperature for 3 h. Frost at 40 C. and lyophilise for 15 h. Dry at 50 C. on P.sub.2O.sub.5 in vacuum oven for 1.5 h.

TABLE-US-00031 Particle size 3-5 um Dry loss 2.1% COOH 19.62% Ca 1.5% Bi 2.2% Na 1.2% Tranexamic acid 1.5% Gelatine 0.32% N 0.11% Aldehyde 0.11%

Example-17. Powder Sample with Calcium (REOXC-Ca) PA-6

[0218] 16.0 g powder REOXC contains 28% COOH, is solved in 3.5 g NaOH in 150 ml demineralised water (solution I). 3.50 g CaCl.sub.2.2H.sub.2O is solved in 20 ml demineralised water and add on solution I. Before mix mechanically for 60 min, add on 4 ml 30% H2O2. pH=3.8-4.0 is adjusted by 20% acetic acid. After adding on 170 ml 98% ethanol, mix at 300 rpm for 30 min. Centrifuge at 4000 rpm for 8-10 min. After rinsing with 60% ethanol three times, mix at 300 rpm for 30 min. Centrifuge at 4000 rpm for 8-10 min. Rinse with 60% ethanol three times and re-centrifuge. Take gel sediment and put petri dishes and frost 45 C. and lyophilise for 24 h. Powder final product is dried at 50 C. on P.sub.2O.sub.5 in vacuum oven.

TABLE-US-00032 Particle size 3-5 um Dry loss 2.1% COOH 20.3% Ca 2.3% Na 0.58% N 0.10% Aldehyde 0.10%

Example-18. Oxidise Cellulose with Antibiotic and Antiseptic (A-REOXC) PA-7

[0219] 10.0 g powder REOXC (20.5-21.5% COOH) mix with 5 g REOXC-Ca,Na and 70 g Tranexamic acid, 0.6 g antibiotic (rifampicin, gatifloxacin, doxycycline, levofloxacin, lincomycin, clindamycin, ciprofloxacin), 0.80 g REOXC-Bi (PA-9), 120 ml 50% ethanol for 3 h. Add on 120 ml isopropanol and mix for 1 h. Centrifuge at 4000 rpm for 10 min. Gel product is put petri dishes and keep at room temperature for 3 h. Frost 40 C. and lyophilise for 18 h. Dry at 50 C. on P.sub.2O.sub.5 in vacuum oven for 1.5 h.

TABLE-US-00033 Particle size 3-5 um Dry loss 2.1% COOH 19.80% Ca 1.8% Bi 1.2% Na 1.2% Antibiotic 3.8% Tranexamic acid 1.7% Aldehyde 0.10%

Example-19. Hemostatic and Anti-Septic Powder with Rifocine as Antibiotic (R-REOXC) PA-8

[0220] 8 g REOXC-Ca,Na mix with tranexamic acid, 0.5 g Rifampicin (2 ampoule rifocine), 0.40 g REOXC-Bi (PA-9), 100 ml 50% ethanol at 300 rpm for 3 h. Add on 120 ml isopropanol and mix for 1.5 h. Centrifuge at 4000 rpm for 10 min. Gel product is put petri dishes and keep at room temperature for 3 h. Frost 40 C. and lyophilise for 18 h. Dry at 50 C. on P.sub.2O.sub.5 in vacuum oven for 1.5 h.

TABLE-US-00034 Particle size 4-6 um Dry loss 2.4% COOH 19.62% Ca 1.8% Bi 1.2% Na 0.88% N 1.11% Rifampicin 4.5% Tranexamic acid 1.7% Aldehyde 0.05%

5. Impregnation of Antibiotics (Rifampicin, Gatifloxacin, Doxycycline, Levofloxacin, Lincomycin, Clindamycin, Ciprofloxacin) to Textile Material

Example-20. Hemostatic, Antiseptic Band with Rifocine as Antibiotic (PA-RB1)

[0221] 0.40 g tranexamic acid mix 0.5 g rifampicin (2 ampoule rifocine), 0.40 g REOXC-Bi (PA-9), 50 ml 50% ethanol at 300 rpm for 3 h. Obtained disperse suspension is impregnated with 10 g REOXC-CaNa (Sample-10). Rinse with 50 ml isopropanol. Use silicone-plastic roller. Wet textile product is put petri dishes and stand at room temperature for 3 h. Frost 45 C. and lyophilise. Dry at 50 C. on P.sub.2O.sub.5 in vacuum oven for 1.5 h. Obtained band is cut and piece together with self adhesive band. Red woven textile is gained. Same process is repeated for other antibiotics (PA-RB, -GB, -DB, -LB, -LIB, CB, CyB)

TABLE-US-00035 Dry loss 2.4% COOH 19.58% Ca 1.8% Bi 1.12% Na 0.85% N 1.11% Rifampicin 4.1% Tranexamic acid 1.0% Aldehyde 0.04%

4.6. Preparation of Hemostatic Gel (GA1-GA9)

Example-21

[0222] 10 g powder (PA1-PA9) is mixed 1 g Chitosan (Deacetyl grade>85%), 1 ml 2% acetic acid, 100 ml 90% ethanol with mechanic stirrer on 350 rpm over night. Disperse mixture centrifuge at 4000 rpm for 10 min. Gel pour petri dishes. of amount is taken by weighing and frost 45 C. Lyophilise for 15 h. Dry at 50 C. on P.sub.2O.sub.5 for 1.5 h.

TABLE-US-00036 Dry loss 11.2% COOH 18.23% Ca 1.62% Na 0.65% Bi 0.92% N 1.25% Tranexamic acid 0.82% Aldehyde 0.03% * Gel is prepared with 2-10 unit weight water according to desired viscosity