FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10Section 10; rule 13)
HYDROGEL COMPOSITION


RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai - 400 701 Maharashtra India.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-


TECHNICAL FIELD
The present invention relates to the hydrogel composition for wound healing. The invention in particular relates to hydrogel compositions comprising PVA and PEG formed by cross-linking a viscous solution of the said composition in the absence of gelling agent. The resulting hydrogel formed in the absence of gelling agent has optimal swelling property and tensile strength.
BACKGROUND ART
Wound dressings are available in the form of hydrogels, hydrocolloids, films, gauzes, alginates, biologies and foams. Amongst these, hydrogels have become more popular because of their relatively high water content, soft and rubbery consistency.
Hydrogels are three dimensional cross-linked polymeric networks that regulate the fluid exchange from the wound surface. Because of their excellent biocompatibility, hydrogels are used in a variety of wound dressings. The three dimensional network of polymers makes them capable of imbibing water 10-1000 times than its original dry weight. Due to its swelling property and water retention capacity, hydrogel finds wide variety of applications in biomedical field ranging from drug delivery system to cell delivery system to wound/burn treatment.
PCT application WO2006125082 provides hydrogel formulation containing pre-solidified hydrogel particles in a precursor hydrogel solution.
Hydrogels have been prepared with various polymers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and polyacrylamides etc.
Various PVA based hydrogels are disclosed in US patent 6231605, 5346935, 5981826, 5522898, 4663358, 4988761. However, in particular, these PVA hydrogels lack the desirable mechanical properties such as sufficient tensile strength and elasticity.
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Polyethylene glycol (PEG) based hydrogels provides a large degree of swelling in aqueous solutions. Various PEG based hydrogels are disclosed in US 5514379, 6362276, and 6541015.
The combined PVA and PEG based hydrogels has been described in PCT WO 2005/120462 which is incorporated herein by reference. This '462 application has described the advantages of combining PEG and PVA polymer. The patent discloses the uses of chemical cross-linkers which are biodegradable groups capped to the PVA and / or PEG to prepare these hydrogels. Once such cross-linkable group of compounds is acrylated glycine anhydride type.
Chemical cross-linkers such as glutaraldehyde and boric acid are used in the preparation of hydrogels. Some of the disadvantages facing such hydrogels include presence of residual cross-linking agents in the hydrogel which could be toxic to the tissues. Usage of such chemicals requires stringent controls during its manufacture which tends to increase the cost of hydrogel manufacture.
On the other hand, preparation of hydrogels using freeze-thaw method does not require the introduction of chemical cross-linking agents. Freeze/thaw cycling of aqueous polymeric solution of PVA results in the formation of physical cross-linking (i.e. weak bonding through a non-permanent "association" of the polymer chains). It is a time-consuming and expensive method. Hydrogels prepared by this method are opaque and this could be a drawback in wound dressing as it prevents visualization and proper examination of the wound bed after its application without the removal of the hydrogel.
Recent attention in preparation of hydrogel wound dressing using gamma irradiation has given the impetus for developing a cost effective hydrogel. One such product that has gained commercial importance is HIZEL™ comprising PVA along with gelling agents as described in Indian Patent Numbers 0187486 and 192136 and WO 2001/030407.
Hydrogels prepared using gamma-irradiation comprises PVA, PVP, PEG acrylamide/maleic acid (CAMA), HPC, etc. as described in following patents
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US Patent No 7235592 describes covalently cross-linked vinyl polymer hydrogel comprising the steps of: providing a physically associated vinyl polymer hydrogel having a crystalline phase; exposing the physically associated vinyl polymer hydrogel to an amount of ionizing radiation providing a radiation dose in the range of about 1-1,000 kGy effective to form covalent crosslinks; and removing physical associations by exposing the irradiated vinyl polymer hydrogel to a temperature above the melting point of the physically associated crystalline phase to produce a covalently cross-linked vinyl polymer hydrogel,
US Patent No 7282165 provides a solution of polyvinyl alcohol in a solvent of DMSO/water. The solution is placed in a mold and is gelated by cycling the mold in a freeze-thaw cycle at a temperature at or below 4.degree. C. for a period of 2 to 24 hours. The hydrogel so formed is washed in a saline solution, including potassium carbonate. The hydrogel is then dehydrated to 20 to 70% water content and thereafter irradiated with Gamma radiation. The surface of the hydrogel is then cross-linked using a boric acid solution preferably between 2.5 and 5% for about 1 minute. The hydrogel is then rinsed and sterilized.
European patent application No. 0107376 (Johnson & Johnson Products Inc.) discloses a wound dressing which is based on a transparent layer of a water-swollen cross-linked homopolymer of N-vinyl pyrrolidone (VP). The homopolymer of VP is first prepared and dissolved in water, a hydrogel layer is then formed by irradiating with gamma rays while contained within a polythene bag,
U.S. Pat. No. 4,989,607, issued to Keusch et al., discloses highly conductive, non-stringy adhesive hydrophilic gels which include, polyvinyl pyrrolidone) cross-linked by radiation to provide the desired non-stringy property thereto.
US Patent No 4871490 provides a method of manufacturing hydrogel dressings from polymers by radiation cross-linking, comprising an aqueous solution containing 2-10 per cent by weight of polyvinylpyrrolidone, no more than 3 percent by weight of agar and 1-3
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percent by weight of poly(ethylene) glycol; pouring the solution into a mould to shape the dressing; tightly closing the mould and subjecting the mould to an ionizing radiation dose in the range of 25-40 KGy.
US Patent No 5401508 provides a hydrogel for ocular comprising acrylamides and acrylates copolymers cross linked by ethylene glycol dimethacrylate
US Patent No 5489437 discloses an adhesive hydrogel product comprising water and a thermoplastic, Hydroxypropyl cellulose (HPC) polymer extrudable in a dry state, then having been exposed to ionizing energy of in excess of 2.5 megarads, and then adding the water, the energy being sufficient to permit cross-linking of the polymer into a tacky hydrophilic hydrogel product having a gel breaking strength of at least 10 p.s.i., and an absorption capacity per mil of thickness in excess of 10%, by weight.
US Patent No 6617372 provides polymeric hydrogel product of polyvinylpyrrolidone
(PVP), poly(vinylcaprolactam) (PVCL), a copolymer of PVP and PVCL and a
comonomer dimethylaminopropyl(meth)acrylarnide (DMAPMA) and/or
dimethylaminoethyl(meth)acrylate (DMAEMA) and a cross linking agent crosslinking agent which is a substantially water-insoluble compound selected from pentaerythritol triallyl ether (PETE) and pentaerythritol tetraacrylate (PETA)., made by irradiating the composition with high energy electron beam or gamma-radiation.
In the above patents, besides the above specified polymers, the hydro gels comprise other components like natural polysaccharides (e.g. Carrageenan, agar etc.) or epichlorhydrin, etc. While some chemicals like epichlorohydrin pose health hazards, natural polysaccharides are prone to microbial contamination thus posing a limitation of its shelf life as well as duration of usage on wounds.
Preparation of hydrogels using radiation technology is generally carried out in the vicinity of the gamma-irradiation unit due to the challenges posed in the transport of free flowing liquid contained in trays. To overcome this, hydrogel compositions generally
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contain gelling agents that allow the polymer solution to set at room temperature that can then be easily transported to irradiation centers for cross-linking the polymer.
Looking into the need for a formulation that would have high viscosity to enable easy transport and at the same time does not include harmful chemicals or gelling agents that might compromise the safety or sterility of the product, the present invention has focused on providing a hydrogel composition which can be shipped to the site of gamma-irradiation unit for crosslinking by increasing the viscosity of PVA and PEG solution without interefering with the swelling capacity of the resulting hydrogel.
The present invention provides a unique combination of PVA and PEG polymer which can be cross-linked by gamma-irradiation. The resulting hydrogel aids in maintaining a moist environment thereby preventing dehydration and scab formation at the wound site and has the ability to imbibe excess wound fluid, thereby preventing maceration. Further, the present invention has developed a hydrogel using calcium chloride as viscosity enhancing agent.
The hydrogel produced by the present invention comprises PVA of low molecular weight (13000 to 23000). The advantage of using low molecular weight PVA versus high molecular weight PVA (available in the public domain) in the preparation of hydrogeis results in hydrogeis exhibiting greater swelling property and elasticity. In high molecular weight hydrogeis, the swelling property and elasticity is compromised due to the greater cross-linking of the polymer..
OBJECTIVE OF THE INVENTION:
It is the aim of the invention to provide a hydrogel composition comprising PVA and PEG which is cross linked by gamma irradiation in the presence of calcium chloride. Calcium chloride increases the viscosity of the PVA and PEG solution thereby ensuring no spillage of the solution during transport of the solution for gamma irradiation. The resulting hydrogel has favorable physical properties such as good swelling property,
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sufficient tensile strength and elasticity that are required during its application on the
wound bed.
It is the aim of the present invention to provide hydrogel composition having lower
molecular weight of PVA in the absence of gelling agent resulting in better tensile
strength
It is the aim of the present invention to provide a hydrogel composition that aids in the transport of the solution without leakage/spillage for gamma-irradiation.
It is the aim of the present invention to provide a hydrogel composition that does not support microbial contamination.
It is the aim of the present invention to provide a hydrogel composition that has enhanced water absorption capacity and good mechanical strength.
It is the aim of the present invention to provide a process for the manufacture of the hydrogel.
It is the aim of the present invention to provide a transparent hydrogel wound dressing.
It is the further aim of the present invention to provide the hydrogel loaded with drugs or biologicals for enhancing its wound healing properties.
SUMMARY OF INVENTION
The present invention provides a hydrogel composition and its methods for preparation. In particular, the present invention discloses the hydrogel comprising PVA and PEG cross-linked by gamma-irradiation using viscosity enhancing agents like calcium chloride
In one embodiment, the present invention provides hydrogel comprising lower molecular weight ranging from 13000 to 23000 PVA, preferably of molecular weight 14,000 PVA.
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In one embodiment, the present invention provides hydrogels comprising PEG molecular weight from 1000 to 4500 preferably PEG of molecular weight 4000.
In one embodiment, the present invention provides hydrogel comprising alkoxy derivatives of PEG as a plasticizer. In one aspect, the present invention employs PEG methyl ether of molecular weight from 750 to 2000. In one preferred aspect, the present invention employs PEG methyl ether of molecular weight of approximately 2,000.
In one embodiment, the present invention provides hydrogel having metal halides such as NaCl, KC1, CaCb, MgCl2 used as such or in combination thereof. Addition of these halides to PVA and PEG solution enhances the viscosity of the solution, thereby facilitating its transport to gamma-irradiation centres. In one aspect, the present invention employs calcium chloride (CaCk)
In one embodiment the present invention provides hydrogel without the use of polysaccharide thus preventing microbial contamination in the resultant composition.
In one embodiment the present invention provides hydrogels that can be administered as various forms such as gels, sheets or gels/sheets impregnated with medicated/non-medicated gauze.
In one embodiment, the present invention provides hydrogels that can be used for all biomedical applications such as wound dressings, medical coatings, skin friendly adhesives, pressure ulcer treatment. In one preferred embodiment, the present invention provides hydrogels for wound care such as diabetic foot ulcers, venous stasis ulcers, pressure ulcers, surgical wounds, ischemic ulcers, traumatic wounds, sores, 1st and 2nd degree burns, abrasions, and lacerations.
In one embodiment, the present invention provides hydrogels that can comprise other drugs or biologicals which will enhance the efficacy of the hydrogel in biomedical applications.
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BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1 demonstrates the optimization and standardization of PVA concentration as well
as thickness for hydrogel formulation.
Figure 2 illustrates the optimization of CaCl2 concentration in hydrogel formulation to set
PVA solution at room temperature
Figure 3 shows the effect of PEG concentration on PVA hydrogels.
Figure 4 illustrates the water absorption capacity of hydrogels.
Figure 5 illustrates the water content of hydrogels.
Figure 6 shows the percentage weight loss of hydrogels as a function of time.
Figure 7 shows the effect of hydrogels on keratinocyte proliferation.
Figure 8 shows the effect of hydrogels on fibroblast proliferation.
DESCRIPTION OF EMBODIMENTS
DEFINITIONS:
The term "hydrogel" as used herein refers to a cross-linked three dimensional network of polymer in aqueous solutions.
The term "PEG" as used herein refers to all polymers of Polyethylene glycol.
The term "PVA" as used herein refers to all polymers of poly (vinyl alcohol).
The present invention provides a hydrogel composition comprising PVA and PEG and its methods of preparation. The hydrogel of the present invention can be used for various biomedical applications.
The PVA used for the hydrogel composition are available from several commercial sources. Generally, increasing the molecular weight of poly (vinyl alcohol) increases the
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tensile strength as well as stiffness. The molecular weight of the PVA used herein is in the range of 13,000-23,000 preferably 14,000. The concentration of PVA used is in the range of 10-20% preferably 20%. A simple cotton gauze as a backing material is impregnated inside the hydrogel to enhance its mechanical properties.
The PEG used for the hydrogel composition are available from several commercial sources. The molecular weight of the PEG used herein is in the range of 1,000-4,500 preferably 4000. The concentration of PEG used is in the range of 0.3-2.0% preferably 0.5%.
The other components used in the hydrogel include PEG methyl ether preferably in the concentration range of 0.1-0.3% and CaCi2 1-10%.
CaCb not only facilitates in cross-linking, it also increases the viscosity of the aqueous PVA solution. It is found that higher the CaCb concentration greater is the viscosity of PVA solution. After dispensing the formulation with calcium chloride into plastic trays, if it is left at room temperature for 12-18 h, it tends to form a plastic sheet. Further, it was observed that the absence of air was critical for efficient-cross linking. Presence of air interferes with cross-linking resulting in incomplete gel formation.
The present invention has focused on addressing the transport conditions required to ship the hydrogel viscous solution to the gamma-irradiation unit.
The process for cross-linking of polymers is preferably by gamma-irradiation. The major advantage of this method is that besides cross-linking, it also is effective in sterilizing the final product. The present invention has studied the effect of the concentration of various components of the hydrogel on the water absorption capacity of the product.
The hydrogel of the present invention has the following features i.e.: 1. It helps in the visualization of the wound-bed without disturbing the hydrogel. Conventional wound dressings require their removal to check the status of the underlying wound bed leading to the disruption of the healing process.
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2. As the hydrogel is non-adhesive, it does not tightly adhere to the wound-bed. During hydrogel dressing change, the hydrogel would leave the wound-bed without disturbing the healing wound.
3. The hydrogel dressing provides a breathable barrier thereby preventing contamination of the underlying wound-bed.
4. It removes the excess liquids and keeps the wound moist. Wound healing is reported to be optimum when the wound is neither dry nor macerated. Presence of water in the hydrogel prevents the drying of the wound bed, while its water absorption property ensures the removal of excess fluids from the wound bed.
5. The said hydrogel is impervious to microbes.
6. By sealing in the natural moisture, the hydrogel helps in maintaining a moist environment, which thereby allows early epithelialization of the wound surface by preventing dehydration and scab formation at the wound site.
7. The presence of gauze aids in easy handling of the hydrogel during its application on the wound. Due to the presence of impregnated-gauze inside the hydrogel, its tensile strength is much higher than that of conventional PVA-based hydrogels.
8. The absence of polysaccharides improves its shelf life.
9. The composition of the present invention is relatively inexpensive due to fewer ingredients.
10. PVA and PEG in the formulation is non-toxic and biocompatible.
The present invention provides hydrogel made as sheets of any size for example: 6cm x 6cm or 10 xlO cm, it can be used as single sheets on small wounds, while multiple sheets can be used on large wounds. Hydrogel sheets can be customized in different shapes and sizes as per the requirement.
The hydrogel dressings is intended to be used as a primary dressing in the management of partial and full-thickness wounds, including diabetic ulcers, venous stasis ulcers, pressure ulcers, surgical wounds, ischemic ulcers, traumatic wounds, superficial burns, donor sites, and in abrasions and lacerations.
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The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: General preparation of Hydrogel:
PVA (20g) was added to 100 ml of de-ionized water and then the mixture was autoclaved for In for complete dissolution. The solution was dispensed into trays and irradiated at 25 kGy for the formation of hydrogels.
A) Effect of the PVA concentration;
The hydrogels of thickness 3, 4 and 5 mm were prepared with 10-20% of PVA. The water absorption capacity of the hydrogel was observed over a period of 96 hours. 20%> of PVA was the optimum concentration and 3 mm was the optimum thickness based on the swelling data. The procedure is mentioned in the section D.
Table 1: Water absorption capacity after 96 hours

Concentration of PVA 3mm 4mm 5mm
20% 227.8 186.22 197.36
17.5 % 154.7 143.72 101.59
15% 106 115.91 112.03
B) Effect of Calcium chloride
An aqueous PVA solution with polymer concentrations from 10 to 20%> was made by adding PVA in CaCfj solution with a concentration range from 1 and 10%> and
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subsequently heating at 121°C. In this case, 0.9 % of NaCl was added to see its influence on water absorption capacity.
Table 2 Optimization of CaCb concentration by water absorption capacity.

Time (h) PVA hydrogel in
1% CaC12 with
NaCl PVA hydrogel in
l%CaC12 without
NaCl PVA hydrogel in
10%CaC12with
NaCl PVA hydrogel in 10%CaC12 without NaCl
0 0 0 0 0
24 130.73 124.33 114.95 108.54
48 140.57 124.99 135.74 121
72 142.08 127.6 139.13 108.61
No significant effect was found in presence of NaCl. As the stickiness increases with increasing CaCl2 concentration, 1% of CaCb with 0.9 % of NaCl was optimized with respect to other parameters as essential characteristics of wound dressing material.
C) Effect of PEG
PEG was used as one of the components in the hydrogel formulation. It was found that in presence of PEG the water absorption capacity of hydrogel increases. With the increase of PEG concentration in hydrogel formulation, water absorption capacity of hydrogel also increases to certain extent. This has been shown in Figure 3.
D) Results
The results of hydrogels with different compositions are enumerated as follows:
a) Study on the water content
The hydrogels were dried at 110°C for 6h to thoroughly remove the water contained therein. The water content of PVA hydrogels were determined from the gel weight W0 and W i, in gram prior to and after drying respectively, by the following formula:
Water content of hydrogel = W0-Wi/W0 * 100
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Table 3 Determination of water content of hydrogels
Sample (s) Initial weight (g) Final weight (g) % Water content
Hydrogel-I 11.75 2.48 78.89
Hydrogel-II 12.95 2.84 78.07
Hydrogel-III 11.86 2.81 76.31
Hydrogel-IV 12.7 2.78 78.11
Hydrogel-V 14.24 3.26 77.11
Hydrogel-I: 20% PVA (by weight)
Hydrogel-II: 20% PVA in 1% CaCl2 (by weight)
Hydrogel-III: 20% PVA in 1% CaCl2 and 0.9% NaCl (by weight)
Hydrogel-IV: 18.02% PVA, 1.8% PEG and 0.18% mPEG (by weight)
Hydrogel-V 18.02% PVA, 1.8% PEG and 0.18% mPEG in 1% CaCl2 (by weight)
b) Study of water absorption capacity of hydrogels
The freshly prepared hydrogel samples were put separately into 10cm x 10cm polystyrene trays containing normal saline solution and were kept in the incubator at 37°C. At predetermined time point, the hydrogels were taken out and the surface water from each hydrogel was removed by cleaning with cotton cloth and the weight of each hydrogel was taken.
The increase in weight was noted and its water absorption capacity was then calculated by using the following formula
Water absorption capacity = WFinal- Wintiail WInitial * 100
Table 4 Determination of water absorption capacity of hydrogels

Time (h) Hydrogel-I Hydrogel-II Hydrogel-III Hydrogel-IV Hydrogel-V
0 0 0 0 0 0
24 98.71 137.72 113.75 228.42 187.91
48 195.97 208.11 212.37 297.41 303.28
72 226.43 237.09 242.91 324.58 354.12
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c) Study on the weight loss
The freshly prepared hydrogel samples were weighed individually and were put separately on each 10 cm x 10 cm glass plate. The hydrogel samples were kept in the oven at 37°C. At predetermined time point, the hydro gels were taken out and were weighed. Percentage weight loss was calculated by the following formula:
% Weight loSS = Winitial - WFina|/ Winitial * 100

Table 5 Del termination of percentage weight loss
Time (h) Hydrogel-I Hydrogel-II Hydrogel-III Hydrogel-IV Hydrogel-V
0 0 0 0 0 0
24 8.11 6.27 8.64 6.5 6.96
48 15.38 14.07 15.4 13.84 13.48
72 23.41 20.18 22.23 21.52 19.42
96 30.94 27.17 28.67 28.86 24.86
120 43.73 40.19 41.63 42.03 27.8
d) Study on water absorption ratio
The hydrogel was dried at 110"C for 6h to remove the water contained therein and the dry weight (Wj) in grams was determined. The dried hydrogel was immersed into freshly prepared normal saline solution and was kept at 37°C. At defined time point, the hydrogels were taken out and their weights (Wt) were measured. This was continued till the hydrogel reached the saturation state. The water absorption ratio was determined as the ratio of the weight (Wt) after absoption of normal saline solution to the dry weight (Wd). Table 6 Weights of the hydrogels at different time intervals

Time (h) Hydrogel-I Hydrogel-II Hydrogel-III Hydrogel-IV
0 2.52 2.81 2.82 2.81
24 26.64 20.98 20.01 26.53
48 33.93 27.09 29.18 39.21
72 35.25 27.19 30.83 43.5
96 35.47 27.07 31.02 45.4
120 33.39 25.22 29.35 44.46
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e) Mechanical properties Tensile strength and Elongation-Tensile strength is performed by elongating a specimen and measuring the load carried by the specimen. From the knowledge of specific dimensions, load and deflection data can be translated into a stress-strain curve. A variety of tensile properties can be extracted from the stress-strain curve.
Tensile strength as well as elongation of the hydrogel is measured using the LLOYD (Model: LRX Plus) instrument at the speed of 500 mm/min maintaining the distance 50 mm between the jaws. In this case, a hydrogel strip with a length of 6 cm and a width of -1.5 cm is cut from 6 cm x 6 cm x 0.3 cm hydrogel, the upper and the lower portion of the hydrogel is wrapped with rough paper and finally, placed in between two clamps.
Table 12 shows that tensile strength of hydrogel increases by impregnating the hydrogel with gauze but percentage elongation decreases as compared to hydrogel without gauze.
Table 12 Data of tensile strength and percentage elongation.

Sample Tensile strength (Kgf/cmA2) Percentage elongation
Hydrogel (with gauze) 12.5 23.51
Hydrogel (without gauze) 0.125 244.51
f) Chemical properties
1. Microbe penetration test
Method:
Bacterial culture lawns (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) were prepared on Blood agar and MacConkey agar plates. Small pieces of hydrogel sheets were placed on the culture lawn. After overnight incubation the upper surfaces of hydrogels were exposed to sterile media contained in petridishes by Agar-
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overlay method. The exposed media in the petridishes were incubated at 37°C for 48
hours and examined for any growth.
Result:
No growth was observed on the exposed media after 48 hrs.
2. Cellular Toxicity
The effect of hydrogel on skin cells were evaluated to ensure that the hydrogels do not release materials that might be detrimental to the growth of skin cells.
For this, the hydrogels were incubated with cell specific culture media for different time points. This contact media was added to cultures of keratinocytes and fibroblasts and its effect on cell growth was monitored for 24h, 48h, 72h and 96h.
Cell proliferation was analysed indirectly by the MTT method. Briefly, MTT (0.5mg/ml) was added to the cells in triplicate dishes after 24, 48, 72 and 96h exposure to the contact media. Viable cells were indirectly determined by their ability to convert soluble MTT to insoluble formazan crystals. The crystals were solubilized and the absorbance was determined as the difference in optical density measured at a test wavelength of 570nm and a reference wavelength of 650nm (Shimadzu UV-VIS Spectrophotometer, Japan). At each end point, the absorbance was recorded and the optical density was compared to control. No toxicity was observed on skin cells incubated with the hydrogel incubated media for defined time periods.
Table 13 Characteristics of hydrogel of the present invention

Properties Hydrogel
% Water absorption capacity (w/w) 300-350
% Water content 75-80%
%Weight loss at 72h 18-20
Thickness 3 mm
Elongation >20%
Tensile strength > 12 Kgf/cm2
Microbial penetration NIL
Cellular toxicity No effect on cell proliferation
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Dated this J day of December, 2008
For Reliance Life Sciences Pvt. Ltd