NOVEL FORMULATION OF PHYSIOLOGICAL CHITOSAN-INORGANIC SALT SOLUTION/BLOOD MIXTURES FOR TISSUE REPAIR
20170333477 · 2017-11-23
Assignee
Inventors
- Wei Ouyang (Montreal, CA)
- Michael Buschmann (Montreal, CA)
- Caroline Hoemann (Montreal, CA)
- Marc Lavertu (Pointe-Claire, CA)
- Anik Chevrier (Point-Claire, CA)
Cpc classification
C08L5/00
CHEMISTRY; METALLURGY
C08L5/08
CHEMISTRY; METALLURGY
C08L5/08
CHEMISTRY; METALLURGY
A61K35/51
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
International classification
A61K35/51
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
A61L27/36
HUMAN NECESSITIES
C08L5/00
CHEMISTRY; METALLURGY
Abstract
The present description relates to a polymer composition for use in repairing tissue of a patient comprising at least one blood component, as polymer, such a chitosan, and at least one inorganic salt, such as NaCl, method of the composition and method of preparing the composition.
Claims
1. A method for repairing a tissue in a subject in need thereof, said method comprising the step of introducing into said tissue a polymer composition, wherein said polymer composition comprises a blood component, a polymer and at least one inorganic salt, wherein said polymer composition is absent a glycerol-phosphate such that the polymer composition adheres to the tissue and promotes cell proliferation for repairing the tissue.
2. The method of claim 1 wherein said composition has a mean coagulation time of less than twelve minutes.
3. The method of claim 1, wherein said tissue is selected from the group consisting of cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, maxillofacial tissues, temporomandibular tissues, abscesses, resected tumors and ulcers.
4. The method of claim 1 wherein said polymer composition is suitable for use as a solidifying implant for repairing the tissue.
5. The method of claim 1 wherein the polymer composition further comprises a mineral acid.
6. The method of claim 1 wherein the polymer is chitosan.
7. The method of claim 1, wherein the at least one inorganic salt is a sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt.
8. The method of claim 1, wherein the at least one inorganic salt is NaCl.
9. The method of claim 1, wherein the blood component is whole blood.
10. The method of claim 1, wherein the composition is a gel.
11. The method of claim 1, wherein the blood component is coagulated.
12. The method of claim 1, wherein the polymer composition has a pH between 6.0 and 7.8.
13. The method of claim 12, wherein the polymer composition has a pH between 6.2 and 6.7.
14. The method of claim 13, wherein the polymer composition has a pH of 6.6.
15. The method of claim 1, wherein the composition has an osmolality between 200 mOsm/kg and 600 mOsm/kg.
16. The method of claim 15, wherein the osmolality is between 326 mOsm/kg and 356 mOsm/kg.
17. The method of claim 15, wherein the osmolality is of 354 mOsm/kg.
18. The method of claim 1, wherein the polymer is chitosan with a degree of deacetylation (DDA) between 20% to 100%.
19. The method of claim 18, wherein the DDA is between 76% and 98%.
20. The method of claim 1, wherein the polymer is chitosan with a number average molecular weight (Mn) between 1 kDa to 10 MDa.
21. The method of claim 19, wherein the Mn is between 2.7 kDa and 298 kDa.
22. The method of claim 5, wherein the mineral acid is hydrochloric acid.
23. The method of claim 1, wherein the blood component to polymer has a ratio of 3:1 v/v.
24. A method of preparing a polymer composition for repairing a tissue in a subject, said method comprising the steps of: a) dissolving chitosan in HCl to provide a chitosan-HCl mixture; b) adding a NaCl solution to the chitosan-HCl mixture to provide a chitosan-HCl—NaCl mixture; and c) admixing at least one blood component to the chitosan-HCl—NaCl mixture to provide the polymer composition.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Reference will now be made to the accompanying drawings.
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DETAILED DESCRIPTION
[0031] It is provided a novel polymer composition comprising a blood component, a polymer and at least one inorganic salt. Related uses and methods are also provided. In an embodiment, such composition can be used for repairing a tissue in a subject. In yet a further embodiment, such composition can be used to limit the time associated with the coagulation of blood in a chitosan composition.
[0032] The polymer composition comprises a polymer, such as, for example chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, keratin sulfate, dermatan sulfate, heparin and heparin sulfate. The polymer should be able to form a gel and be used for the treatment of tissue repair.
[0033] The polymer composition also comprises a blood component. Any blood component is contemplated herein, such as whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood or placenta blood. In an embodiment, the blood component is derived from whole blood to be enriched or depleted for a specific blood component. In a preferred embodiment, the mix ratio between the blood component and the polymer is about 3:1
[0034] The polymer composition also comprises at least one inorganic salt. In a preferred embodiment, NaCl is used. Alternatively, any inorganic salt, including sodium, chloride, potassium, calcium, magnesium, phosphate, sulfate, carboxylate salt, such as KCl, CsCl, CaCl.sub.2, CsF, KClO.sub.4, NaNO.sub.3 and CaSO.sub.4 are also contemplated herein.
[0035] The polymer composition can further comprise an acid, such as a mineral acid or an organic acid. The acid is used to lower the pH of the composition to facilitate dissolution of the chitosan. In a preferred embodiment, HCl is used as the acid. Alternatively, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid or hydrobromic acid could also be used.
[0036] In another embodiment, the polymer composition consists essentially of a blood component, a polymer and at least one inorganic salt. Such polymer composition does not contain additional components which participates to the formation of the gel or coagulation of the blood (such as coagulation products for example) but can contain other components such as an acid (to facilitate the dissolution of the chitosan), preservatives, etc.
[0037] It is specifically described herein the formulation of chitosan and a blood component, under physiological conditions (pH, osmolality). Briefly chitosan-HCl—NaCl solutions are mixed with blood, to create blood/chitosan-HCl—NaCl. The blood/chitosan-HCl—NaCl compositions are capable of forming a gel. In addition, the blood component in the blood/chitosan-HCl—NaCl is capable of coagulating to form an implant for tissue repair. In addition, when chitosan having a number average molecular weight lower than M.sub.n 232 kDa is used, the chitosan-HCl—NaCl solutions mixed with blood described herein coagulates faster than chitosan-HCl-βGP solutions.
[0038] A preferred embodiment is shown in Example 1 where a novel formulation of physiological chitosan-HCl—NaCl solution was prepared with chitosan M.sub.n 75 kDa; 79% DDA and mixed with whole blood. Blood/chitosan-HCl—NaCl mixtures coagulated faster than blood/chitosan-HCl-βGP mixtures to create fast-coagulating blood/chitosan implants for tissue repair.
[0039] Chitosan (1.62% w/w)-HCl (38 mM)-NaCl (160 mM) solution at pH of 6.50 and osmolality of 354 mOsm/kg, and chitosan (1.62% w/w)-HCl (71 mM)-PGP (2.15% w/w) solution at pH of 6.65 and osmolality of 334 mOsm/kg (Table 1) were mixed with fresh rabbit whole blood. To prepare chitosan/blood clots, immediately following collection of whole blood, a volume of 900 μl of whole blood was mixed into a cryotube containing 300 μl of 1.62% chitosan-HCl—NaCl solution (or 1.62% chitosan-HCl-βGP solution) and three 0.39 g stainless steel balls. The mixture was shaken by hand for 10 seconds (the mix ratio of blood to chitosan was 3 to 1).
[0040] 250 μl was transferred into 3 glass tubes at 37° C. with a 1 ml syringe to prepare 3 clots (implants). One clot was used to test coagulation time and fixed immediately after it coagulated, the second clot was used to test coagulation time and fixed at 60 minutes after mixing, and the third clot was used to test coagulation time and mechanical strength after 60 minutes.
[0041] Coagulation time results (Tables 2-5 and
[0042] After 60 minutes, some serum exuded from the clots (Table 6). The homogeneity evaluation results are shown in Table 7.
[0043] Another embodiment is shown in Example 2 where chitosans of different molecular weightsM.sub.n were used to prepare a novel formulation of physiological chitosan-HCl—NaCl mixtures (Table 8). Blood/chitosan-HCl—NaCl mixtures coagulated faster than blood/chitosan-HCl-βGP mixtures when the chitosan has a number average molecular weight M.sub.n<232 kDa to create fast-coagulating blood/chitosan-HCl—NaCl implants with good mechanical strength for tissue repair (Tables 9-12 and
[0044] After 60 minutes, some serum exuded from the clots and the homogeneity evaluation results are shown in Table 17.
[0045] It is thus demonstrated herein that blood/chitosan-HCl—NaCl mixtures coagulated faster than blood/chitosan-GP mixtures when the chitosan had a molecular weight M.sub.n<232 kDa to create blood/chitosan clots (implants) with good mechanical properties. Chitosans with a number average molecular weightM.sub.n>232 kDa were also used to prepare blood/chitosan-HCl—NaCl mixtures that coagulated to create blood/chitosan clots (implants) with good mechanical properties. Therefore, these solutions can be alternatives to the current chitosan-HCl-βGP solution known in the art with the advantage of solidifying faster.
[0046] The compositions described herein can be used to improve the repair and to regenerate cartilaginous tissues and other tissues including without limitation meniscus, ligament, tendon, bone, skin, comea, periodontal tissues, abscesses, resected tumors, and ulcers.
[0047] There is also contemplated herein the use of the polymer compositions described herein that can be placed or injected into a body site where the mixture aids the repair, regeneration, reconstruction or bulking of tissues. Repaired tissues include for example without limitation cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors, and ulcers. The tissue that can be repaired or regenerated is for example without limitation cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissue, abscesses, resected tumors, or ulcers. In some cases, the site of introduction in the body may be surgically prepared to remove abnormal tissues. Such procedure can be done by piercing, abrading or drilling into adjacent tissue regions or vascularized regions to create channels for the polymer composition to migrate into the site requiring repair.
[0048] The present disclosure will be more readily understood by referring to the following examples which are given to illustrate embodiments rather than to limit its scope.
Example 1
Formulation and Characterization of Fresh Blood/Physiological Chitosan Solution Mixtures (Rabbit Blood)
[0049] 1—Preparation of Chitosan (1.62% w/w)-HCl (38 mM)-NaCl (160 mM) Solution; without NaOH, pH: 6.6; Total Volume: 10.0 ml
[0050] 0.180 g of chitosan (degree of deacetylation or DDA=79% and number average weight or M.sub.n75 kDa) was weighted in a 20 ml beaker. H.sub.2Odd was added to the beaker until the weight of chitosan+H.sub.2O=9.34 g. A magnetic stir bar was added into the beaker and the solution was stirred for about 10 minutes in order to hydrate the chitosan powder as much as possible. 0.38 ml of HCl 1 N (Sigma, Product No 318949) was added to the solution under moderate stirring. The beaker was covered with Parafilm™, and the solution was heated to about 60° C. for 2 hours, stirred overnight until completely dissolved. 0.32 ml of 5N NaCl (Sigma, Product No S-9888) solution was added into the beaker and mixed. The obtained pH of the chitosan solution was physiological at 6.5 and the osmolality was also physiological at 354 mOsm/kg (Table 1).
2—Preparation of Chltosan(1.62% w/w)-HCl (71 mM)-SGP (2.15%) Solution, pH: 6.6, Total Volume: 9.0 ml
[0051] 0.162 g of chitosan (degree of deacetylation or DDA=79% and number average weight or M.sub.n=75 kDa) was weighted in a 20 ml beaker. H.sub.2Odd was added to the beaker until the weight of chitosan+H.sub.2O=6.65 g. A magnetic stir bar was added into the beaker to stir the solution for about 10 minutes in order to hydrate the chitosan powder as much as possible. 0.55 ml of HCl 1 N (Sigma, Product No 318949) was added to the solution under moderate stirring. The beaker was covered with Parafilm™ and stirred overnight until completely dissolved. 1.8 ml of 10.75% pGP (Sigma, Product No G9891) in 50 mM HCl solution was added into the beaker and mixed (mix ratio of chitosan solution/PGP solution is 4:1). The obtained pH of the chitosan solution was physiological at 6.7 and the osmolality was also physiological at 334 mOsm/kg (Table 1).
TABLE-US-00001 TABLE 1 Composition and properties of chitosan solutions. Solution No and C.sub.chitosan C.sub.HCl C.sub.NaCl C.sub.βGP pH Osmolality type. (% w/w) (mM) (mM) (% w/w) Precipitation (Measured) (mOsm/kg) Chitosan- 1.62 38 160 — no 6.50 354 HCl—NaCl pH 6.6 Chitosan- 1.62 71 — 2.15 no 6.65 334 HCl-βGP pH 6.6
3—Drawing Blood
[0052] Blood was extracted from rabbits using sterile technique, starting by injecting 0.3 cc/kg Hypnorm® IM to the rabbits (for example 0.9 cc for a 3 kg rabbit). First, for each rabbit, ˜2 ml of blood was collected in a Vacutainer® tube containing EDTA (Fisher, BD, Product No 02-683-99A) to obtain CBC (complete blood count) and platelet count. Second, for each rabbit, ˜5 mL of blood was collected using a sterile 5 cc syringe (Fisher, BD, Product No 309604). Four rabbits were used in this experiment.
4—Preparing Blood/Physiological Chitosan Solution Mixtures, Measuring the Coagulation Time and Mechanical Strength of the Clots
[0053] To prepare blood/chitosan-HCl—NaCl (pH 6.6) clots-1, immediately following collection of whole blood, a 900 μl pipet of whole blood was added into a cryotube containing three 0.39 g stainless steel balls and 300 μl chitosan solution and mixed by hand shaking for 10 seconds. 250 μl was then transferred into 3 glass tubes at 37° C. with a 1 ml syringe to prepare 3 clots: the first clot was used to test coagulation time and fixed immediately after it coagulated; the second clot was used to test coagulation time and fixed at 60 minutes after mixing; the third clot was used to test coagulation time and mechanical strength after 60 minutes. This experiment was accomplished in triplicate for each rabbit. To prepare blood/chitosan-HCl-βGP (pH 6.6) clots-2, the same procedure was repeated as described hereinabove, but a chitosan-HCl-βGP (pH 6.6) solution was mixed with blood.
[0054] Coagulation was determined by visualization of the clot at 37° C. All the three glass tubes were used for testing coagulation time. The glass tubes were gently taken from the hot plate vertically every minute, slowly tilted, and the blood mixture was visualized at the bottom of tube. If the mixture was immobile and formed clot, it was coagulated; if the mixture was still mobile at the bottom of the tube, it was not coagulated yet. Mechanical strength was tested by putting the clot on the centre of the palm and pressing the clot with a finger until it was crushed. The resistance to compression, liquid expression and crushed appearance were also observed. The mechanical strength was scored with a 4 “+” system: “+” represents clot was easily broken and crushed appearance was multiple fragments (more than 5 fragments); “++” represents clot was relatively firm and crushed appearance was multiple fragments (3-5 fragments); “+++” represents clot was firm and elastic, crushed appearance was 2-3 fragments: “++++” represents clot was firm and elastic, crushed appearance was 2 fragments (sometimes still connected) or there was just a hole in the center of clot.
[0055] For Rabbit 259F (R1F), all the blood/chitosan mixtures coagulated within 10 minutes (from 7 minutes to 10 minutes) and formed firm elastic clots (see Table 2). The coagulation time of blood/chitosan-HCl—NaCl pH 6.6 mixtures (8 minutes) was significantly less than the coagulation time of blood/chitosan-HCl-PGP pH 6.6 mixtures (9.7 minutes) (
TABLE-US-00002 TABLE 2 Coagulation time of blood/chitosan clots from rabbit R259F (R1F). Coagulation Mean Sample time value Mixture number (Min) (Min Fresh Mixture 1 1 8 8 blood/chitosan- 2 8 HCl—NaCl pH 6.6 3 8 (clot1) Mixture 2 1 9 2 9 3 9 Mixture 3 1 7 2 7 3 7 Fresh Mixture 1 1 10 9.7 blood/chitosan- 2 10 HCl- βGP pH 6.6 3 10 (clot2) Mixture 2 1 9 2 9 3 9 Mixture 3 1 10 2 10 3 10
[0056] For Rabbit 260F (R2F), all the blood/chitosan mixtures coagulated within 9 minutes and formed firm elastic clots (see Table 3 and
TABLE-US-00003 TABLE 3 Coagulation time of blood/chitosan clots from rabbit R260F (R2F) Coagulation Mean Sample time value Mixture number (Min) (Min) Fresh Mixture 1 1 9 8.3 blood/chitosan- 2 9 HCl—NaCl pH 6.6 3 9 (clot1) Mixture 2 1 8 2 8 3 8 Mixture 3 1 8 2 8 3 8 Fresh Mixture 1 1 9 8.7 blood/chitosan- 2 9 HCl- βGP pH 6.6 3 9 (clot2) Mixture 2 1 9 2 9 3 9 Mixture 3 1 8 2 8 3 8
[0057] For Rabbit 261M (R3M), all the blood/chitosan mixtures coagulated within 15 minutes (from 6 minutes to 15 minutes) and formed firm elastic clots (see Table 4). The coagulation time of blood/chitosan-HCl—NaCl pH 6.6 mixtures (7.3 minutes) was significantly less than the coagulation time of blood/chitosan-HCl-βGP pH 6.6 mixtures (14.3 minutes) (
TABLE-US-00004 TABLE 4 Coagulation time from rabbit R261M (R3M) Coagulation Mean Sample time value Mixture number (Min) (Min) Fresh Mixture 1 1 6 7.3 blood/chitosan- 2 6 HCl—NaCl pH 6.6 3 6 (clot1) Mixture 2 1 8 2 8 3 8 Mixture 3 1 8 2 8 3 8 Fresh Mixture 1 1 14 14.3 blood/chitosan- 2 14 HCl-βGP pH 6.6 3 14 (clot2) Mixture 2 1 15 2 15 3 15 Mixture 3 1 14 2 14 3 14
[0058] For Rabbit 262M (R3MR4M), all the blood/chitosan mixtures coagulated within 14 minutes (from 7 minutes to 14 minutes) and formed firm elastic clots (see Table 5). The coagulation time of blood/chitosan-HCl—NaCl pH 6.6 mixtures (8.3 minutes) was significantly less than the coagulation time of blood/chitosan-HCl-βGP pH 6.6 mixtures (12.7 minutes) (
TABLE-US-00005 TABLE 5 Coagulation time from rabbit R262M (R4M) Coagulation Mean Sample time value Mixture number (Min) (Min) Fresh Mixture 1 1 7 8.3 blood/chitosan- 2 7 HCl—NaCl pH 6.6 3 7 (clot1) Mixture 2 1 8 2 8 3 8 Mixture 3 1 10 2 10 3 10 Fresh Mixture 1 1 12 12.7 blood/chitosan- 2 12 HCl-βGP pH 6.6 3 12 (clot2) Mixture 2 1 12 2 12 3 12 Mixture 3 1 14 2 14 3 14
[0059] The mechanical strength results (Table 6 and
TABLE-US-00006 TABLE 6 Mechanical strength test of blood/chitosan clots. Rabbit Mixture Sample Resistance to Liquid Crushed No. component No. compression expressed appearance R259F Fresh Clot1-1 Firm and elastic Some liquid exuded Break into 2 fragments but blood/chitosan- ++++ ++ still connected HCl—NaCl pH 6.6 Clot1-2 Firm and elastic Some liquid exuded Break into 2 fragments but (clot1) ++++ ++ still connected Clot1-3 Firm and elastic Some liquid exuded Break into 2 fragments but ++++ ++ still connected Fresh Clot2-1 Firm and elastic Some liquid exuded Not broken but a hole in the blood/chitosan- ++++ ++ center of clot HCl- βGP pH 6.6 Clot2-2 Firm and elastic Some liquid exuded Not broken but a hole in the (clot2) ++++ ++ center of clot Clot2-3 Firm and elastic Some liquid exuded Break into 2 fragments but ++++ ++ still connected R260F Fresh Clot1-1 Firm and elastic Some liquid exuded Break into 2 fragments but blood/chitosan- ++++ ++ still connected HCl—NaCl pH 6.6 Clot1-2 Firm and elastic Some liquid exuded Break into 2 fragments but (clot1) ++++ ++ still connected Clot1-3 Firm and elastic Some liquid exuded Break into 2 fragments but ++++ ++ still connected Fresh Clot2-1 Firm and elastic Some liquid exuded Not broken but a hole in the blood/chitosan- ++++ ++ center of clot HCl- βGP pH 6.6 Clot2-2 Firm and elastic Some liquid exuded Not broken but a hole in the (clot2) ++++ ++ center of clot Clot2-3 Firm and elastic Some liquid exuded Not broken but a hole in the ++++ ++ center of clot R261M Fresh Clot1-1 Firm and elastic Some liquid exuded Not broken but a hole in the blood/chitosan- ++++ ++ center of clot HCl—NaCl pH 6.6 Clot1-2 Firm and elastic Some liquid exuded Not broken but a hole in the (clot1) ++++ ++ center of clot Clot1-3 Firm and elastic Some liquid exuded Break into 2 fragments but ++++ ++ still connected Fresh Clot2-1 Firm and elastic Some liquid exuded Not broken but a hole in the blood/chitosan- ++++ ++ center of clot HCl- βGP pH 6.6 Clot2-2 Firm and elastic Some liquid exuded Not broken but a hole in the (clot2) ++++ ++ center of clot Clot2-3 Firm and elastic Some liquid exuded Not broken but a hole in the ++++ ++ center of clot R262M Fresh Clot1-1 Firm and elastic Some liquid exuded Break into 2 fragments but blood/chitosan- ++++ ++ still connected HCl—NaCl pH 6.6 Clot1-2 Firm and elastic Some liquid exuded Not broken but a hole in the (clot1) ++++ +++ center of clot Clot1-3 Firm and elastic Some liquid exuded Break into 2 fragments but ++++ + still connected Fresh Clot2-1 Firm and elastic Some liquid exuded Not broken but a hole in the blood/chitosan- ++++ ++ center of clot HCl- βGP pH 6.6 Clot2-2 Firm and elastic Some liquid exuded Not broken but a hole in the (clot2) ++++ ++ center of clot Clot2-3 Firm and elastic Some liquid exuded Break into 2 fragments but ++++ ++ still connected
5—Histological Treatment and Homogeneity Evaluation of Blood/Chitosan Clots
[0060] Fixed clots were sectioned in two parts. One part was cryoprotected with sucrose, infiltrated with OCT, cryosectioned, stained with Safranin O/Fast Green and observed by optical microscopy. One part was stored at 4° C. until further use. All the photos were taken from the blood/chitosan clot samples fixed 60 minutes after the clots were prepared; two photos taken with 5× and 40× objectives from different regions of each sample were used for homogeneity evaluation. Each specimen was observed under microscopy with special emphasis on: presence of bubbles or cracks; presence and distribution of precipitates of chitosan described as: large aggregates or small aggregates; chitosan distribution and whether or not they are homogeneously dispersed across the section; erythrocyte morphology in terms of discoid, shrunken, swollen or chaining.
[0061] Histology showed that the homogeneity of clots prepared with βGP was better (8 of 12 samples scored as “+” and none scored as “−”) than the homogeneity of the clots prepared with NaCl (3 of 11 samples scored as “+” and 3 of 11 samples scored as “−”) (Table 7).
TABLE-US-00007 TABLE 7 Homogeneity of blood/chitosan clots. Clot-1 Clot-2 sample Homogeneity of sample Homogeneity of (with NaCl) clot-1 (with βGP) clot-2 FB 1-1-R259F + FB 2-1-R259F + FB 1-2-R259F + FB 2-2-R259F + FB 1-3-R259F − FB 2-3-R259F ± FB 1-1-R260F + FB 2-1-R260F + FB 1-2-R260F ± FB 2-2-R260F + FB 1-3-R260F − FB 2-3-R260F + FB 1-1-R261M ± FB 2-1-R261M ± FB 1-2-R261M − FB 2-2-R261M + FB 1-3-R261M * FB 2-3-R261M + FB 1-1-R262M ± FB 2-1-R262M + FB 1-2-R262M ± FB 2-2-R262M ± FB 1-3-R262M + FB 2-3-R262M ± * data not available
Example 2
Formulation and Characterization of Fresh Blood/Chitosan Clots by Using Chitosan Solutions with Different Molecular Weight (Rabbit Blood)
[0062] 1—Preparation of Chitosan (1.62% w/w)-HCl (38 mM)-NaCl (160 mM) Solution; without NaOH, pH: 6.6; Total Volume: 10.0 ml
[0063] The chitosan-HCl—NaCl solutions were prepared as described previously with chitosans of different M.sub.n and DDA (lot No. AS-144-02-A: M.sub.n 2.7 kDa and DDA 98%; lot No. CH10075: M.sub.n 75 kDa and DDA 79%; lot No. CH0100702B: M.sub.n 232 kDa, and DDA 81%; lot No. CH0050602A: M.sub.n 298 kDa and DDA 76%). The pH of the chitosan solutions were physiological (6.2 to 6.7) and the osmolality was also physiological (326 to 356 mOsm/kg) (see Table 8).
2—Preparation of Chitosan (1.62% w/w)-HCl (71 mM)-βGP (2.15%) Solution, pH: 6.6, Total Volume: 9.0 ml
[0064] The chitosan-HCl—NaCl solutions were prepared as described previously with chitosans of different M.sub.n and DDA (lot No. AS-144-02-A: M.sub.n 2.7 kDa and DDA 98%; lot No. CH10075: M.sub.n 75 kDa and DDA 79%; lot No. CH0100702B: M.sub.n 232 kDa, and DDA 81%; lot No. CH0050602A: M.sub.n 298 kDa and DDA 76%). The pH of the chitosan solutions were physiological at 6.7 and the osmolality was also physiological (340 to 345 mOsm/kg) (Table 8).
TABLE-US-00008 TABLE 8 Composition and properties of chitosan solutions C.sub.chitosan C.sub.HCl C.sub.NaCl C.sub.βGP pH Osmolarity Solution No and type. (% w/w) (mM) (mM) (% w/w) Precipitation (Measured) (mOsm) Chitosan-HCl—NaCl 1.62 55 160 no no 6.19 356 pH 6.6 (AS-144-02-A) Chitosan-HCl-βGP 1.62 77 No 2.15 no 6.69 345 pH 6.6 (AS-144-02-A) Chitosan-HCl—NaCl 1.62 38 160 no no 6.62 326 pH 6.6 (CH10075) Chitosan-HCl-βGP 1.62 71 No 2.15 no 6.67 344 pH 6.6 (CH10075) Chitosan-HCl—NaCl 1.62 38 160 no no 6.65 339 pH 6.6 (CH0100702B) Chitosan-HCl-βGP 1.62 71 No 2.15 no 6.71 337 pH 6.6 (CH0100702B) Chitosan-HCl—NaCl 1.62 38 160 no no 6.58 332 pH 6.6 (CH0050602A) Chitosan-HCl-βGP 1.62 71 No 2.15 no 6.68 340 pH 6.6 (CH0050602A)
3—Drawing Blood
[0065] Blood was extracted from rabbits using sterile technique, as described previously. Four rabbits were used in this experiment.
4—Preparing Blood/Physiological Chitosan Solution Mixtures, Measuring the Coagulation Time and Mechanical Strength of the Clots
[0066] Rabbit whole blood/chitosan-HCl—NaCl mixtures and rabbit whole blood/chitosan-HCl-βGP mixtures were prepared at a mix ratio of 3:1 v/v as described previously. This experiment was accomplished in triplicate for each rabbit. Coagulation of the clot was determined by visualization of the clot at 37° C. as described previously. Mechanical strength was tested as described previously.
[0067] For chitosan lot No. AS-144-02-A (M.sub.n of 2.7 kDa and 98% DDA), all the blood/chitosan mixtures coagulated within 18 minutes (from 10 minutes to 18 minutes) and formed firm elastic clots (see Table 9). The coagulation time of blood/chitosan-HCl—NaCl pH 6.6 mixtures was significantly less than the coagulation time of blood/chitosan-HCl-PGP pH 6.6 mixtures (
TABLE-US-00009 TABLE 9 Coagulation time of fresh blood/chitosan mixtures (Lot No. AS-144-02-A) Concentration of Coagulation Mean chitosan solution time value Mixture and mixing ratio (Min) (Min) Comments Fresh blood/ 1.62% and 3:1 1 12 11.3 All the mix- Chitosan- (clot1-1-1) 2 11 ture samples HCl—NaCl 3 11 coagulated 1.62% and 3:1 1 11 11 within 12 (clot1-1-2) 2 11 minutes (from 3 11 10 minutes to 1.62% and 3:1 1 11 10.7 12 minutes). (clot1-1-3) 2 10 3 11 Fresh blood/ 1.62% and 3:1 1 18 17.3 All the mix- Chitosan- (clot1-2-1) 2 17 ture samples HCl- βGP 3 17 coagulated 1.62% and 3:1 1 17 16.7 within 18 (clot1-2-2) 2 16 minutes (from 3 17 15 minutes to 1.62% and 3:1 1 15 15.3 18 minutes). (clot1-2-3) 2 15 3 16
TABLE-US-00010 TABLE 10 Mechanical strength test of fresh blood/chitosan clots (Lot No. AS-144-02-A) Resistance to Liquid Crushed Sample compression expressed appearance Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl + clot1-1-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl + clot1-1-2 (1.62%, 3:1) Fresh blood/ Firm and elastic A few liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl ++ clot1-1-3 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl- βGP + clot1-2-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot1-2-2 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl- βGP + clot1-2-3 (1.62%, 3:1)
[0068] For chitosan lot No. CH10075 (M.sub.n of 75 kDa and 79% DDA), all the blood/chitosan mixtures coagulated within 13 minutes (from 6 minutes to 13 minutes) and formed firm elastic clots (Table 11). The coagulation time of blood/chitosan-HCl—NaCl pH 6.6 mixtures was significantly less than the coagulation time of blood/chitosan-HCl-pGP pH 6.6 mixtures (
TABLE-US-00011 TABLE 11 Coagulation time of fresh blood/chitosan mixtures (Lot No. CH10075) Concentration of Coagulation Mean chitosan solution time value Mixture and mixing ratio (Min) (Min) Comments Fresh blood/ 1.62% and 3:1 1 7 7 All the mix- Chitosan- (clot2-1-1) 2 7 ture samples HCl—NaCl 3 7 coagulated 1.62% and 3:1 1 6 6.3 within 7 (clot2-1-2) 2 7 minutes (from 3 6 6 minutes to 1.62% and 3:1 1 6 6 7 minutes). (clot2-1-3) 2 6 3 6 Fresh blood/ 1.62% and 3:1 1 12 12.3 All the mix- Chitosan- (clot2-2-1) 2 12 ture samples HCl- βGP 3 13 coagulated 1.62% and 3:1 1 13 12.7 within 13 (clot2-2-2) 2 13 minutes (from 3 12 12 minutes to 1.62% and 3:1 1 13 12.7 13 minutes). (clot2-2-3) 2 12 3 13
TABLE-US-00012 TABLE 12 Mechanical test of fresh blood/chitosan clots (Lot No. CH10075) Resistance to Liquid Crushed Sample compression expressed appearance Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl—NaCl + clot2-1-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl + clot2-1-2 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl + clot2-1-3 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot2-2-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl- βGP + clot2-2-2 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot2-2-3 (1.62%, 3:1)
[0069] For chitosan lot No. CH0100702B (M.sub.n of 232 kDa and 81% DDA), all the blood/chitosan mixtures coagulated within 7 minutes (from 3 minutes to 7 minutes) and formed firm elastic clots (see Table 13 and
TABLE-US-00013 TABLE 13 Coagulation time of fresh blood/chitosan mixture (Lot No. CH0100702B) Concentration of Coagulation Mean chitosan solution time value Mixture and mixing ratio (Min) (Min) Comments Fresh blood/ 1.62% and 3:1 1 7 6.7 All the mix- Chitosan- (clot3-1-1) 2 7 ture samples HCl—NaCl 3 6 coagulated 1.62% and 3:1 1 7 6.7 within 7 (clot3-1-2) 2 7 minutes (from 3 6 6 minutes to 1.62% and 3:1 1 7 6.3 7 minutes). (clot3-1-3) 2 6 3 6 Fresh blood/ 1.62% and 3:1 1 3 3.7 All the mix- Chitosan- (clot3-2-1) 2 4 ture samples HCl- βGP 3 4 coagulated 1.62% and 3:1 1 4 3.3 within 4 (clot3-2-2) 2 3 minutes (from 3 3 3 minutes to 1.62% and 3:1 1 3 3.3 4 minutes). (clot3-2-3) 2 3 3 4
TABLE-US-00014 TABLE 14 Mechanical test of fresh blood/chitosan clots (Lot No. CH0100702B) Resistance to Liquid Crushed Sample compression expressed appearance Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl + clot3-1-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl ++ clot3-1-2 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl—NaCl + clot3-1-3 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP ++ clot3-2-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot3-2-2 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot3-2-3 (1.62%, 3:1)
[0070] For chitosan lot No. CH0050602A (M.sub.n of 298 kDa, 76% DDA), all the blood/chitosan mixtures coagulated within 7 minutes (from 5 minutes to 7 minutes) and formed firm elastic clots (see Table 15 and
TABLE-US-00015 TABLE 15 Coagulation time of fresh blood/chitosan mixture (Lot No. CH0050602A) Concentration of Coagulation Mean chitosan solution time value Mixture and mixing ratio (Min) (Min) Comments Fresh blood/ 1.62% and 3:1 1 7 7 All the mix- Chitosan- (clot4-1-1) 2 7 ture samples HCl—NaCl 3 7 coagulated 1.62% and 3:1 1 7 6.7 within 7 (clot4-1-2) 2 7 minutes (from 3 6 6 minutes to 1.62% and 3:1 1 7 6.3 7 minutes). (clot4-1-3) 2 6 3 6 Fresh blood/ 1.62% and 3:1 1 7 6.7 All the mix- Chitosan- (clot4-2-1) 2 6 ture samples HCl- βGP 3 7 coagulated 1.62% and 3:1 1 7 6.7 within 7 (clot4-2-2) 2 7 minutes (from 3 6 5 minutes to 1.62% and 3:1 1 6 5.7 7 minutes). (clot4-2-3) 2 6 3 5
TABLE-US-00016 TABLE 16 Mechanical test of fresh blood/chitosan clots (Lot No. CH0050602A) Resistance to Liquid Crushed Sample compression expressed appearance Fresh blood/ Firm and elastic Almost no liquid 2 fragments Chitosan- ++++ expressed HCl—NaCl + clot4-1-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl—NaCl + clot4-1-2 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl—NaCl + clot4-1-3 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid Hole in center Chitosan- ++++ expressed HCl- βGP + clot4-2-1 (1.62%, 3:1) Fresh blood/ Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot4-2-2 (1.62%, 3:1) Fresh blood/- Firm and elastic Almost no liquid 2 fragments but Chitosan- ++++ expressed still connected HCl- βGP + clot4-2-3 (1.62%, 3:1)
5—Histological Treatment and Homogeneity Evaluation of Blood/Chitosan Clots
[0071] Histological treatment and homogeneity evaluation was performed as described hereinabove.
[0072] Histology showed that the homogeneity of clots prepared with RGP was better (9 of 12 samples scored as “+” and none scored as “−”) than the homogeneity of the clots prepared with NaCl (2 of 12 samples scored as “+” and 7 of 12 samples scored as “−”) (see Table 17).
TABLE-US-00017 TABLE 17 Homogeneity of blood/chitosan clots Homogeneity Homogeneity Clot samples of clots Clot samples of clots (with NaCl) (with NaCl) (with βGP) (with βGP) blood 1-1-1 + (between ± blood 1-2-1 ± (AS-144-02-A) and +) (AS-144-02-A) blood 1-1-2 ± (between ± blood 1-2-2 + (between ± (AS-144-02-A) and +) (AS-144-02-A) and +) blood 1-1-3 ± (between ± blood 1-2-3 + (between ± (AS-144-02-A) and +) (AS-144-02-A) and +) blood 2-1-1 − blood 2-2-1 + (CH10075) (CH10075) blood 2-1-2 − blood 2-2-2 + (between ± (CH10075) (CH10075) and +) blood 2-1-3 − blood 2-2-3 + (CH10075) (CH10075) blood 3-1-1 + blood 3-2-1 + (CH0100702B) (CH0100702B) blood 3-1-2 + blood 3-2-2 + (CH0100702B) (CH0100702B) blood 3-1-3 − blood 3-2-3 + (CH0100702B) (CH0100702B) blood 4-1-1 − blood 4-2-1 + (CH0050602A) (CH0050602A) blood 4-1-2 − blood 4-2-2 + (CH0050602A) (CH0050602A) blood 4-1-3 − blood 4-2-3 + (CH0050602A) (CH0050602A)
[0073] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.