Claims:1) A medical dressing comprising chitosan powder /flakes, water and acetic acid.
2) The medical dressing as claimed in claim 1, wherein the medical dressing is porous and in form of sponge.
3) The medical dressing as claimed in claim 1, wherein pore size of the medical dressing is ranging from about 30 µm to 200 µm.
4) The medical dressing as claimed in claim 1, wherein moisture content of the medical dressing is less than about 2%.
5) The medical dressing as claimed in claim 1, wherein the medical dressing comprises at least about 98% chitosan.
6) The medical dressing as claimed in claim 1, wherein polydispersity index of the chitosan is about 1.
7) The medical dressing as claimed in claim 1, wherein degree of deacetylation of chitosan is greater than about 85%.
8) The medical dressing as claimed in claim 1, wherein absorption efficiency of the medical dressing is ranging from about 20% to 540%.
9) The medical dressing as claimed in claim 1, wherein time taken for complete haemostasis by the medical dressing is less than about 240 seconds. , Description:This invention relates to medical dressing and the method of preparation thereof.
In present invention medical dressing for wound care, especially for treatment of haemorrhage, burns, tissue regeneration is prepared by using controlled lyophilisation of chitosan.
Background of invention:
Several advancements has been made in the area of wound care in last few decades, however pre-hospital wound care and continuous wound care are still underserved globally. Bleeding to death from traumatic injury is common in civilian and battlefields. This could may or may not be accompanied with burns and other wounds that could be acute in nature.
Accidents and trauma related injuries account for approximately 10% of deaths per year. Uncontrolled bleeding from wound sites is a major cause of these preventable deaths. From a fatal wound approximately 40ml/min of blood is lost and if it continues for 20 minutes, the victim dies of hemorrhagic shock. Bleeding to death before reaching medical facility is common to battlefield injuries as well. Very often victims bleed to death due to lack of pre-hospital care, time and distance to reach a medical facility. Due to the absence of external haemostatic products, the primary intervention to stop bleeding still remains to be cotton gauze using pressure. Lack of adequate pre-hospital care (golden hour) is stated as one of the prominent reasons of trauma related deaths.
Transportation of burn victim to the nearest medical facility is also a major unsolved issue. Due to this victims die of post injury infections which are preventable deaths. Though there is an unmet need in both the cases, it is pertinent to note that there is not even a single product which can stop external bleeding quickly or to protect burn wounds until the victim reaches a medical facility.
At present the related products in market are not made to address all these issues, but they have limited success to solve specific problems. With advent of certain novel biomaterials coupled with advanced bio-engineering processes, better alternative and efficient solutions in wound care are now emerging. Availability from natural sources and properties like non-immunogenicity, biocompatibility, charge density, biodegradability, mucoadhesiveness and no risk of viral infections makes them desirable for various biomedical applications. These characteristics make them ideal candidates to be used in tissue engineering, drug delivery, permeable membrane systems, tissue sealants etc.
There has been plenty of works have been done in this field. U.S. Patent No.: 7,482,503 B2 relates to the process to make wound dressing formed of biomaterial comprising chitosan for controlling severe bleeding which involves formation of aqueous solution of chitosan, placement of this solution in the mold, freezing of this solution in the mold and then removal of the water from frozen chitosan structure by a prescribed freeze-drying process to form sponge chitosan structure, after this compression of sponge chitosan structure by the application of heat and pressure to reduce the thickness and increase the density.
U.S. Pat No.: 6,998509 B1 relates to the process to make wound care device comprising chitosan, said chitosan being capable of absorbing liquid to form a swollen, coherent gel, and said chitosan being in the form of fibers having been modified by treatment with acid in a solvent which is not able to dissolve the chitosan fibers and by treatment with heat.
The application of heat in above stated inventions may destroy the integrity of structure of biomaterial. The above stated inventions also fail to address the issue of batch to batch variability of chitosan that affects the end product, which is a major problem associated with natural polymers. (Alam H. B., Burris D., Dacorta J. A. & Rhee P., Hemorrhage Control in the Battlefield: Role of New Hemostatic Agents. MILITARY MEDICINE 2005, 170:63-69)
U.S. Patent Publication No.: US 2007/0237811 A1 describe a composition in which chitosan is prepared in a foamed gel that may be layered onto a suitable backing for use as a wound dressing, or the gel may be directly applied to wound to effect hemostatic activity as a result of action of the chitosan.
Patent Publication No.: CN 1364646 (A) describes the medical dressing includes chitosan, acetic acid and medical enzyme, preferably FE complex enzyme. But the usage of enzymes is not economical at industrial level.
Considering the aforesaid problems, a simple, rapid and relatively inexpensive medical dressing is required that can prevent haemorrhage, prevent microbial infections, protect burn wounds and also aid in tissue regeneration.
Objects of the Invention:
The main object of the invention is to provide multi-utility natural medical dressing that can prevent haemorrhage, prevent microbial infections, protect burn wounds and also aid in tissue regeneration.
Another object of this invention is the preparation of a medical dressing using controlled lyophilisation of chitosan.
Further object of this invention is to maintain the structural integrity of the biomaterial by avoiding the usage of heat and employing the lyophilisation process in which the water removal of frozen biomaterial is done by passing the chitosan solution in solid frozen phase into a gas phase without the substantial formation of an intermediate liquid phase.
It is an additional object of this invention is the development of a quick freezing cycle (of less than 5 hours) for the optimal formation of pores with adequate robustness which also helps to reduce the entire lyophilisation cycle to 25 hours.
It is still another object of this invention is to address the raw material batch variability by keeping polydispersity index around 1 and thus making the biomaterial consistent throughout the batches.
Summary of invention
The present invention relates to multi-utility medical dressing that can prevent haemorrhage, prevent microbial infections, protect burn wounds and also aid in tissue regeneration and a process for preparation of it. In the present invention poly [ß-(1, 4)-2-amino-2-deoxy-D-glucosamine] commonly known as chitosan, glacial acetic acid and deionized water are used. A process for producing this medical dressing comprises preparation of aqueous chitosan solution and addition of glacial acetic acid in it; after that mixing of solution to achieve dissolution and complete homogenization of it; filtering it; lyophilizing with in specially designed metal molds to produce a porous, flexible, interconnected dressing; and then cutting the stabilised end product, vacuum packing of it and finally sterilizing it by gamma irradiation up to 15kGy.
Brief Description of Drawing
Figure 1: Shows medical dressing.
Figure 2a: Shows image of medical dressing under Scanning Electron Microscope at 1mm magnification.
Figure 2b: Shows image of medical dressing under Scanning Electron Microscope at 200 µm magnification.
Figure 3: Shows Mechanism of action of medical dressing onto the wound.
Figure 4: Shows the flow chart of manufacturing process outline of medical dressings.
Detailed description
The nature of the invention and the manner in which it is performed is clearly described in the specification. The invention has various components and they are clearly described in the specification.
In the present invention poly [ß-(1, 4)-2-amino-2-deoxy-D-glucosamine] commonly known as chitosan, glacial acetic acid and deionized water are used. Besides chitosan other active biomaterials such as CMC, alginates, hyaluronic acid, chitin, carageenan and silk fibroin can be used for the purpose of this invention.
Chitosan (poly [ß-(1, 4)-2-amino-2-deoxy-D-glucosamine]) a natural biomaterial extracted from crustaceans is a linear polysaccharide which is structurally similar to cellulose and poly cationic in nature. The amino group in poly [ß-(1, 4)-2-amino-2-deoxy-D-glucosamine] is positively charged and this charge density is dependent on pH and the degree of deacetylation value. It is insoluble in water or in alkaline solutions at pH levels above about 6.5, or in organic solvents. It is highly bioadhesive and readily binds to negatively charged surfaces such as mucosal membranes. It also enhances the transport of polar drugs across epithelial surfaces, and is biocompatible and biodegradable. Chitosan has been proven to do work as hemostatic and chelating agents. It is a biocompatible material and that’s why can be used to replace or repair any body tissues or bodily function. Chitosan is an effective cellular agglutinating agent. The antimicrobial properties of chitosan have been reported against number of microorganisms and these properties are due to the strong positive charge of the chitosan molecule and thus it acts as a coagulant of the microbial cells. Wound dressing scaffolds that are made from high molecular weight natural biomaterials like chitosan with least polydispersity are fabricated into 2D scaffolds with interconnected pores.
Glacial acetic acid (CH3COOH), also known as ethanoic acid, is a pure, water-free organic acid that is colourless with pungent odour. Glacial acetic acid is hygroscopic meaning it absorbs water from the environment (www.mixmedicine.com/aceticacid.htm visited on February 27, 2010).
Deionized water is a type of purified water with mineral ions (salts) removed. These mineral ions include sodium, calcium, iron, copper, chloride, and bromide. Deionized water is created by taking conventional water and exposing it to electrically charged resins that attract and bind to the salts, removing them from the water (www.wisegeek.com/what-is-deionized-water.htm visited on February 27, 2010).
The process for preparation of medical dressing comprises of following steps:
1. Aqueous chitosan solution is prepared by dispersing dry chitosan powder/flakes in de-ionized water. Chitosan is obtained from Primex EHF, Iceland.
2. Glacial acetic acid is then added drop by drop to the aqueous mixture.
3. Mixing of the above stated solution is done at 200 rpm using a mechanical stirrer at room temperature for 5 hours to achieve the dissolution and complete homogenization.
4. The filtration is done by pouring the solution onto the filter and made to pass through it.
5. The filtrate is collected in the glass beaker at room temperature.
6. Lyophilisation:
a. Aqueous chitosan solutions are poured into trays.
b. Trays are loaded to the lyophilizer chamber.
c. Then the solution is passed through lyophilizing cycle.
8. Stabilised end products are individually packed in laminated metal pouches which are then vacuum sealed.
9. Individually packed final products are then terminally sterilised using gamma irradiation.
Lyophilizing cycle parameters are as follows:
a. Aqueous chitosan solution is loaded at 10 °C ± 2 °C.
b. After that freezing is done at 10 °C ± 2 °C for 20 min ± 10 min.
c. Then temperature is reduced to 5 °C ± 2 °C for 10 min ± 5 min.
d. Temperature at 5 °C ± 2 °C is maintained for another 1 hour ± 10 min.
e. Then temperature is reduced to -15°C ± 2 °C for 20 min ± 10 min.
f. This reduced temperature is maintained for another 1 hour ± 10 min.
g. Again temperature is reduced to -30°C ± 2 °C.
h. This reduced temperature is maintained for 2 hours ± 10 min.
i. Then evacuation is done by creating vacuum of 300 µbar.
j. Chitosan solution in frozen phase is then subjected to drying at -15 °C ± 2 °C creating vacuum at 265 µbar for 40 min ± 10 min.
k. This drying is maintained for another 3 hour ± 10 min at -15 °C ± 2 °C and 265 µbar vacuum.
l. Then drying is done at 0 °C ± 2 °C temperature and 227 µbar vacuum for 20 min ± 10 min.
m. This drying is again maintained at 0 °C ± 2 °C temperature and 227 µbar vacuum for another 3 hours ± 10 min.
n. Then drying is done by increasing temperature up to the 15 °C ± 2 °C at 165µbar vacuum for 20 min ± 10 min.
o. This temperature is maintained at 15 °C ± 2 °C and 165µbar vacuum for another 5 hours ± 10 min.
p. Then drying is done at 30 °C ± 2 °C and 133µbar vacuum for 20 min ± 10 min.
q. This temperature is maintained at 30 °C ± 2 °C and 133µbar vacuum for 6 hours ± 10 min.
The formulation consists of 1% to 4% (w/v) of poly [ß-(1,4)-2-amino-2-deoxy-D-glucosamine], 0.5% to 2%(v/v) glacial acetic acid and de-ionized water (Q.S.).
The examples describe and demonstrate embodiments within the scope of the present invention. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope.
Example:
Ingredients Amount
Poly [ß-(1,4)-2-amino-2-deoxy-D-glucosamine] 2% (w/v)
glacial acetic acid 1 % (v/v)
Deionised water Q.S.

The process for preparation of medical dressing comprises of following steps:
1. Aqueous chitosan solution is prepared by dispersing dry chitosan/flakes in de-ionized water in clean, sterile, 500mL Borosilicate flasks.
2. Glacial Acetic acid is then added drop by drop to the aqueous mixture.
3. Mixing of the above stated solution is done at 200 rpm using a mechanical stirrer at room temperature for 5 hours to achieve the dissolution and complete homogenization.
4. The filtration is carried out by using 100 mesh metal sieves. The solution is poured onto the filter and made to pass through it.
5. The filtrate is collected in the glass beaker at room temperature.
6. Lyophilisation:
a. Aqueous chitosan solutions are poured into Teflon coated SS 316 trays of the dimension 25cm X 30cm X 0.5cm using Peristaltic pump.
b. Trays are loaded to the chamber of BOC Edwards 20kg lyophilizer.
c. Then the solution is passed through lyophilizing cycle.
d. Stabilised end products are individually packed in steel pouches which are then vacuum sealed.
e. Individually packed final products are then terminally sterilised using C60 gamma irradiation load of up to 15kGy which would ensure a SAL of 10-6.
Tests conducted
1) Product characterization tests:
Parameters Process Results
Moisture content
Subjected to Hot air oven until constant weight achieved Less than 2%
Pore size
determination
Scanning electron microscopy at electron beam of 30KV at 0.8 Torr and GSE detector 30 µm to 200µm
Viscosity
Brooke field viscometer LV3 Spindle and temperature below 35°C 50-500cPs
Degree of Deacetylation
Fourier Transform Infra-red spectroscopy
More than 85%

Interpretation: though the moisture content was obtained less then 2%, the pore size was 30 µm to 200µm, viscosity was 50-500cPs and degree of deacetylation was more than 85%, it full-fills the acceptability criteria
2) Hemostatic efficiency test:
Evaluation of the hemostatic efficiency test was done on albino rabbits. Natural medical dressing was tested on a comparative study on accepted animal models of hemorrhage with HemCon® hemostatic bandage (manufactured by HemCon® MEDICAL TECHNOLOGIES INC) taken as control. For the studies 6 healthy, adult rabbits were selected. The animals were acclimatized under controlled environment for 5 days. The hematological parameters of the animals were checked and monitored.
The following parameters were evaluated for the study:
• experiment Time taken for complete haemostasis
• Survival of animal and body weight
• Observation of re-bleeding
• Time taken for re-bleeding
• Absorption of blood by the dressing
• Animal blood pressure after
Experimental design:
Details Test material Control material
No. of animals 6
Name of species Rabbit Rabbit
Name of strain Albino Albino
Sex of animals Male/female Male/female
Body weight 2.5kg 2.5kg
Route of application Application of the material as such in wound generated in marginal ear artery Application of the material as such in wound generated in marginal ear artery
Surface area/ amount of material required 3 x 3cm2 material of known weight 3x 3cm2 material of known weight
Procedure: General anesthesia with a mixture of ketamine and xylazine was administered to 6 healthy, adult rabbits (2kg to 3kg). The rabbits were shaved and disinfected on both ears. Using a longitudinal incision, 1cm wound segments were made on the right and left marginal ear arteries. Through the resultant jet spray of blood, the right wound was immediately covered with 2 x 2 cm2 piece of the Medical dressing, of known weight W1. Similarly left wound was covered with commercially available bandage of known weight W2 .Direct pressure was applied for 2 min and then samples were removed and weighed immediately, W3 for Medical dressing with blood and W4 for commercially available HemCon® bandage with blood.
Observation Tables:
A) Blood absorption data:
Animal ID Weight of the material Weight of the material with blood Blood absorbed
Test
(Medical dressing) W1(gm) Control (HemCon®)
W2(gm) Test
(Medical dressing) W3(gm) Control
(HemCon®)
W4(gm) Test
(Medical dressing)
W1-W3 (gm) Control
(HemCon®) W2-W4 (gm)
255 0.1653 0.2876 0.2454 0.4074 0.0801 0.1198
232 0.1945 0.2888 0.4969 0.3600 0.3024 0.0712
206 0.1590 0.3680 0.3078 0.4540 0.1488 0.0860
212 0.1767 0.3452 0.3781 0.3982 0.2014 0.0530
249 0.2042 0.2987 0.2450 0.3713 0.0408 0.0726
247 0.1577 0.3167 1.0100 0.3623 0.8523 0.0456
Mean 0.1762 0.3175 0.4472 0.3922 0.271 0.0747
Average absorption efficiency(in percent) % absorption= final weight – initial weight ÷ initial weight×100 153% 23%

Results: The results of the study shown in observation table indicate that the weight of the medical dressing was increased 153% while the weight of HemCon® was increased 23% that means medical dressing was absorbed 130% more blood as compared to the HemCon® bandage.
B) Observations for Medical dressing & Control (HemCon® bandage):
Animal ID/No:
255 232 206 212 249 247
Health of animal Normal Normal Normal Normal Normal Normal
Number of vessels lacerated
Medical dressing 1 1 1 1 1 1
HemCon® 1 1 1 1 1 1
Time taken for haemostasis
HemCon® 2min 2min 2min 2min 2min 2min
Medical dressing 2min 2min 2min 2min 2min 2min
Pre-treatment blood loss
Medical dressing Nil Nil Nil Nil 1 drop Nil
HemCon® Nil Nil 3 drops Nil Nil Nil
Observation of Re-bleeding
Medical dressing Nil Nil Nil 6min later 11 min later Nil
HemCon® 60 min later Nil Nil Nil Nil Nil
Time taken for re-bleeding (duration)
Medical dressing Nil Nil Nil 30sec 60sec Nil
HemCon® 30sec Nil Nil Nil Nil Nil
Time taken for complete hemostatsis
Medical dressing 195sec 210sec 240sec 240 sec 180 sec 180 sec
HemCon® 770sec 600sec 660 sec 300 sec 270 sec 180 sec
Survival of the animal
Medical dressing Yes Yes Yes Yes Yes Yes
HemCon® Yes Yes Yes Yes Yes Yes

Results: The results of the study shown in observation table suggest that re-bleeding in 3 animals was occurred. The average time taken for complete hemostasis by medical dressing was 208 sec and that by HemCon® bandage was 463 seconds. The results indicate that the hemostatic efficiency of Medical dressing is higher when compared to the HemCon® bandage.
3) Test for invitro Cytotoxicity
Method of analysis: Test on extracts based on ISO 10993-5, 1999
Summary of the process: Extract was prepared by incubating the test material with physiological saline at 37 ±2°C for 24-26 h and diluted with medium containing serum to get an extraction ratio of 1.25cm2/ml The extract was incubated after dilution with L-929 cell lines for about 24-26 h and examined periodically. Along with this phenol was used as positive control and high density poly ethylene as negative control.
Sr. No Sample Cytotoxicity Scale Interpretation
1 Negative control
0 Non cytotoxic
2 Positive control 4 Severe cytotoxic
3 Medical dressing 0 Non cytotoxic

Results: The results suggest that the extract did not show any Cytotoxicity just at that of negative control and the positive control showed severe Cytotoxicity. The following is the qualitative evaluation on basis of microscopic examination.
4) Test for Irritation test (skin)
Method of analysis: ISO10993-10:2002/Amd: 1 2006(E) Medical Evaluation of medical devices: Part 10. Test for irritation and delayed type hypersentivity: clause 6.3. Animal skin irritation test.
Summary of the process: The test material (Medical dressing) moistened with physiological saline and applied topically on upper left side of the animal. Along with test material a sterile gauze piece moistened with physiological saline applied on the lower side of the animal. The application sites were covered and observed at 1h, 24h, 48h and 72h after removal of patches of evidence of any tissue reaction. Grading of the tissue reaction of erythema and oedema was done at each observation period. The primary irritation index was calculated by adding the scores of each animal and dividing by the total number of animals.
Observation Table:
Sr.no Animal No Type of reaction Observation period
24 h 48h 72h
1 186 Erythema Test site: 0
Control site: 0 Test site: 0
Control site: 0 Test site: 0
Control site: 0
Oedema Test site: 0
Control site: 0 Test site: 0
Control site: 0 Test site: 0
Control site: 0
2. 215 Erythema Test site: 0
Control site: 0 Test site: 0
Control site: 0 Test site: 0
Control site: 0
Oedema Test site: 0
Control site: 0 Test site: 0
Control site: 0 Test site: 0
Control site: 0
3 208 Erythema Test site: 0
Control site: 0 Test site: 0
Control site: 0 Test site: 0
Control site: 0
Oedema Test site: 0
Control site: 0 Test site: 0
Control site: 0 Test site: 0
Control site: 0

Irritation score:
Sr no. Animal no Erythema Oedema
1 186 0 0
2 215 0 0
3 208 0 0

Result: The requirements of the tests are met if the primary irritation index is 0.4 or less. The results indicated that the medical dressing did not produce any irritation following the direct application on skin.
Substantial work has been undertaken to determine an optimum lyophilisation cycle which is crucial to the medical material preparation. Freezing step during lyophilisation is one of the most critical steps, as the preferred structure of scaffold is formed during the same. This particular step determines the porosity, interconnected pore structure, flexibility and natural robustness to the medical material. Lyophilisation process employs a quick freezing, long drying process that enhances medical dressing porosity and flexibility. In the present invention usage of heat is avoided and it employs the lyophilisation process in which the water removal of frozen biomaterial is done by passing the chitosan solution in solid frozen phase into a gas phase without the substantial formation of an intermediate liquid phase, which maintains the structural integrity of the biomaterial. This invention also employs the quick freezing cycle (of less than 5 hours) for the optimal formation of pores with adequate robustness which also helps to reduce the entire lyophilisation cycle to 25 hours.
For some natural polymers PDI is almost taken as unity. But, it is a manufacturing challenge to keep it as unity due to uneven chain length distribution. In this invention Chitosan with polydispersity index of 1 is used and thus it mitigates the batch variability of the end product. As batch variability is a major performance issue with the predicate products, this approach helps us to produce large scale less variable dressings.