Title of Invention

A PROCESS FOR THE PREPARATION OF RECONSTITUTED COLLAGEN SOPONGE

Abstract

A process for the preparation of reconstituted collagen sponge by adding the collagen free from non-collagenous materials to 0.2 to 5-1% w/v of foaming agent selected from surfactants, 0.5 - 1% v/v cross linking agent and 1 - 5% v/v pharmaceutically and pharmacologically active component, lyophilizing the resulting mixture by conventional method, sterilizing the lyohilised product by conventional method to obtain reconstituted collagen sponge.

Full Text

The present invention relates to a process for the preparation of reconstituted collagen sponge. More particularly, the present invention relates to a process for the preparation of reconstituted collagen sponge useful as a biomaterial for medical applications. The reconstituted collagen sponge of the present invention has potential use as wound dressing aid, bone-cartilage substitute, surgical tampons, laparatomy pads and a container for drug release. The product serves as a better scaffolding for re- epithelialization or good granulation of tissue to ensure faster recovery of wound as well as relief from pain. It prevents excessive evaporation of fluid from the wound ensuring minimum fluid loss from the wound site and also prevents any infection, thereby enhancing healing of wounds, burns, excision wounds etc. Collagen, a ubiquitous protein of mammalian bodies, is especially useful in biomedical applications because of its compatibility with body tissues and fluids. As reported by Chvapil (Biology of Collagen, Ed. Vidik and Vuust, Academic Press. London, p-313,1980), even though the use of collagen as a biomaterial dates back to the end of the nineteenth century, it is only recently that the advance of collagen chemistry has facilitated the development of highly sophisticated collagenous biomedical products. Physical properties of collagen with reference to the high tensile strength as well as the fibrillar nature of the fibers allow further orientation, weaving, or knitting of this protein. As reported by Prockop and Kivirikko (Annual Review of Biochemistry, 64, 403, 1995) and Chvapil ( Biology of collagen, Edited by Viidik. and Vuust, Academic Press, London, p-313, 1980), type I collagen has extensively been used to formulate medical materials, primarily because of its abundant availability in nature and its unique physico-chemical as well as biological properties. The ability of collagen to crosslink with a variety of agents allows a controlled and predictable change of solubility, swelling and rate of degradation of this protein. Besides high tensile strength, such properties as the ability to be cross-linked and the affinity towards various cells have projected collagen as the biomaterial of choice for a number of important medical applications. Collagen biomaterials are approved by the US Food and Drug Administration (FDA) for using in surgery as well as injectable implant materials. The protein has a safety profile as a biomaterial and can be produced in forms that are easily used in minimally invasive procedures. A good biomaterial for use in surgery should be strong, pliable, elastic amenable to sterilisation and relatively inert biologically and chemically. When used as a dressing material it should be able to prevent infection from air borne bacteria, stop flow of body fluids from the wound and help in healing and formation of uniform granulation tissue. When used internally it should be amenable for absorption in the living system without causing oedema, allergic reactions and autogenic reactions. It should be available in sterile ready to use form, should have reasonable shelf life and should be made available at reasonable cost. As reported by Chvapil (Journal of Biomedical Material Research, 11, 721,1977), native collagen is usually non toxic, but massive dose of it is likely to provoke antigenic reactions in animal body. Thus the antigenic character of collagen is of prime importance while considering this protein as a biomaterial and this has been a matter of interest among the researchers. Seller and Reynolds (Biochemical Journal,.167, 353,1977), Timple and Gay (Journal of Immunological Methods, 18, 165,1977), have observed that the antigenic tendencies can even be practically removed by the elimination of telopeptides. Natural collagen, as it exists in tendon, skin or other collagen rich tissue, exhibits resistance to biological degradation. But the product, which is either regenerated from dispersions or reconstituted from solutions will not exhibit resistance to biological degradation, resulting in handling problems, when implanted in animal tissues and is generally not directly applicable for use in medical devices. This limitation can be overcome by suitably crosslinking the collagen preparations. As reported by Chvapil and Holusa (Journal of Biomedical Material Research, 2, 245,1968), highly cross-linked collagen, used for covering tissue surfaces to absorb fluids as well as to prevent drying, does not promote any immunological reaction. As reported by Houck and Chang (Proceedings of the Society of Experimental Biology and Medicine, 138, 342, 1971), the large surface of collagen fibrils attract fibrinogenic cells and even the break down products of collagen are chemotactic, thereby contributing to wound healing. Another possible role of exogenous collagen in wound healing, as observed by Wood (Biochemical Journal, 84, 429,1962), is that certain neutral salt molecules cause soluble collagen to act as nucleation centres, forming fibrillar structures resulting in wound healing. Despite a long history in soft tissue repair applications, only a limited number of collagen based devices specific to wound care are commercially available. As reported by Chvapil etal (International Review of Connective Tissue Research, 6, 1, 1973), collagen rich tissues have not been used in medicine as such with a few exceptions such as surgical sutures from submucosa of sheep intestine, serosa of beef intestine, rat tail tendon etc. The reason behind this is the existence of collagen in tissues along with various non collagenous structures that will modify otherwise minimal antigenicity of this protein and affect several other parameters such as ion-exchange function, reactivity with cells and biodegradability. Several ingenious methods were, therefore developed to prepare extremely pure collagen either in the form of a solution or gel or pure fibril arrangement. As reported by Cavarallo et al (Biotechnology and Bioengineering, 43, 781, 1994) and Meade and Silver (Biomaterials, 11, 176, 1990), in these methods, the collagenous tissues are solubilised by dilute acids, buffers or alkalis. The pure collagen is obtained by successive treatments using salts and enzymes to remove non collagenous impurities. The major limitation of all these methods is that the solubilisation of the collagen tissues results in degradation of the soft collagenous tissues. The hitherto known processes for making soluble collagen involve the extraction of soluble collagen, from tendons and other collagenous tissues, yielding both soluble and insoluble extracts, whereby the insoluble extract is rejected using only the soluble extract further for the preparation of type I collagen. Thus these processes are very much uneconomical, especially while using achilles tendons, which are very difficult to solubilise. Collagen can be used as an effective biomaterial by removing the non-collagenous materials, thereby strengthening the remaining collagen material. Palmieri (International Journal of Tissue Reactions, 21, 14, 1992) and Zitelli (Advances in Dermatology, 243, 2, 1987) have reported that collagen matrices were found to adhere well to wounds, absorb many times their weight in fluids and are effective in supporting new tissue growth. Collagen matrices have been designed in the form of solid articles such as tubes, sponges and threads for use as blood vessels, burn dressings. Collagen heamostatic agents are used with greater sophistication in procedures involving gynaecology oral surgery, laporoscopy etc. Collagen sheet is a temporary biological cover for denuded areas of body surface due to 1st, 2nd and 3rd degree non-infected or infected burns, trauma, chronic skin ulcers, skin donor sites and amputation sites etc. Collagen sheets are derived from bovine tissues. The sheets consist of mainly type I collagen with small percentage of type n and type V collagens. The sheets are commercially available in India. According to Grant and Jackson (Essays in Biochemistry, 12, 77, 1976), the collagen sponges and fleece are shown to have good incorporating characters that are suited for wound care. Collagen sponge has an edge, due to its porosity in structure, over the other preparations in providing a scaffold for tissue in growth, thereby resulting in the increase of fibroblasts biosynthesis, ensuring the quickening of the healing process of animal dermal wounds. Collagen sponges present an added benefit for soft tissue repair, because their wet strength that allows for suturing the material to soft tissue, provide a template for new tissue in growth. Grille and Gross (Journal of Surgical Research, 2, 69 1962), Grant and Jackson (Essays in Biochemistry, 12, 77, 1976), have reported that foetal calf skin collagen of the bovine animal has been the raw material of choice, while preparing collagen sponge by the hitherto known processes due to its easy availability from the slaughtered animals. Moreover, the foetal calf skin is reported to have a very similar structural parameters to type 1 collagen of human being. Meade and Silver (Biomaterials, 11, 176, 1990), have prepared collagen sponge for the treatment of ulcers, tendon repair and nerve regeneration. The sponges were prepared by adding lyophilised collagen to distilled water in a blender and the pH was adjusted to 3.0 using concentrated hydrochloric acid. The mixture was blended at increasing speeds (up to 1000 rpm) until it was viscous. Collagen dispersion was then de-aerated in a side-armed flask under house vacuum for 1 h and then freeze-dried in a metal pan to a final thickness of about 2 mm. Drewek et al (Biomaterials, 17, 1733, 1996), have prepared collagen sponge containing gentamicin for investigating drug delivery profile of collagen implants. Bovine skin is the starting material for collagen sponges containing gentamicin. After liming and removal of the epidermis and subcutis, it is diced. The cube-shaped pieces are then cleaned, delipidated and dried. The resulting collagen granulate is suspended in water for injections containing gentamicin at the required concentration and homogenized. Aliquots of the suspension containing the drug are placed in glass or aluminium containers, frozen and then freeze-dried. The lyophilizate is then packed and sterilized. Mizuno and Glowacki (Biomaterials, 17, 1819, 1996) have also prepared collagen sponge for implanting demineralized bone powder in muscle to stimulate chondrogenisis followed by bone formation. Pepsin digested bovine collagen solutions are diluted with acetic acid and neutralised with buffer and sodium bicarbonate. These collagen solutions are poured into specially designed moulds of tygon tubing and frozen at -20 ° C. The sponges thus obtained are irradiated by UV light. The major limitation of the hitherto known processes is that the non-collagenous particles, associated with the collagenous source, are not removed by the conventional acid treatment, thereby resulting in impurities in the final product, rendering the process uneconomical from commercial point of view. Moreover, the presence of the non collagenous particles are likely to cause immune reactions in the system while using it as an implant material. According to Chvapil (Journal of Biomedical Material Research, 11, 721, 1977) surfactants have bactericidal and sperimicidal effects and also show beneficial effect on fluid binding capacity of any biomaterial. Hence the addition of surfactant in the sponge preparation, makes the sponge a better biomaterial. The main objective of the present invention is to provide a process for the preparation of reconstituted collagen sponge from collagenous source for medical applications. Another objective of the present invention is to remove the non-collagenous particles associated with the collagenous source. Yet another objective of the present invention is to achieve pure soluble collagen from the available collagen tissues. Still another objective of the present invention is to ensure that the collagen primary structure and its properties remain intact in the sponge. Yet another objective of the present invention is to protect the triple helix structure of the collagen. Still another objective of the present invention is to produce the collagen free from non collagenous impurities. Accordingly the present invention provides a process for the preparation of reconstituted collagen sponge which comprises i) adding the collagen free from non-collagenous materials to 0.2 to 5-1% w/v of foaming agent selected from surfactants, 0.5 - 1% v/v cross linking agent such as herein described and 1 - 5% v/v pharmaceutically and pharmacologically active component selected from antibiotics, biocides, antitumour drugs, growth regulators ii) lyophilizing the resulting mixture, as formed in step (i), by conventional method, iii) sterilizing the lyohilised product, as formed in step (ii), by conventional method to obtain reconstituted collagen sponge. ii. lyophilising and sterilising the lyophilised product, by conventional method to obtain reconstituted collagen sponge. In an embodiment of the present invention the foaming agent used may be such as Ethylene oxide condensate of nonyl phenol, Sodium lauryl sulfate, Isooctyl phenoxy poly ethoxy ethanol In still another embodiment of the present invention, the amount of the foaming agent used may be preferably in the range of 0. 5-0.75% by weight on the volume of the collagen solution. In still another embodiment of the present invention, the cross linking agents used may be such as Glutaraldehyde, Hexamethylene di isocyanate. In yet another embodiment of the present invention, the pharmacologically and physiologically active components used may be such as antitumor drugs, growth regulators and antibiotics In yet another embodiment of the present invention, the method used for sterilisation of the reconstituted collagen sponge may be such as gamma ray irradiation, ethylene oxide treatment. The collagen used in this invention may be prepared in the following manner A source of collagenous tissue is washed well in water and chopped into smaller pieces, which are minced at 10-20° C. The minced material is then scoured using min. 0.2% w/w of a surfactant, on the weight of the minced collagenous tissue, at a temperature of max. 40° C. The scoured mass is then slimed with min.O. 2% of sliming agent, at 30-40° C. The slimed mass is then washed thoroughly to make the same free from non-collagenous particles, fat and other chemicals. The resulting stock is treated with min. 0.5-2% w/w, of a proteolytic enzyme on the weight of the minced collagenous tissue at 2-8° C for 12-48 hours. The pH of the bath is adjusted in the range of 2-3.5. The enzyme treated stock is then homogenised using a conventional homogeniser at a temperature of max. 37° C and diluted with 150 - 400% v/v, water to form a collagen solution. The viscous solution, thus formed, is treated with min. 5% of a precipitant on the volume of the collagen solution with continuous stirring. The resulting suspension is separated conventionally by centrifuging in the range of 10000 - 20000 rpm at 4-8° C and the precipitated collagen is dissolved in 200-400% v/v of an acid at a pH in the range of 2- 4. The homogenous solution, thus obtained, was dialysed against 5 lit of 0.02 M di sodium hydrogen phosphate solution. The dialysate was centrifuged at 10000 rpm and the precipitate was redissolved in 500 ml of 0.5M acetic acid and dialysed against 5 Its of distilled water for 10-48 hours. The pure viscous soluble collagen thus obtained is taken in a beaker. A mixture containing 0.25 - 1% vv/v, of a foaming agent , 0.5 - 1% v/v, of a crosslinking agent and 1-5% v/v, of the pharmacologically active compound is then added to it with vigorous stirring over a period of 30-120 minutes till maximum froth is produced. The resulting mass with froth is then frozen immediately in nitrogen atmosphere and is then lyophilised at -60° C and 80 Torr to get reconstituted collagen sponge, which is stored in an air tight container at a temperature of maximum 25° C. The lyophilisation brought about a great increase in the internal surface area in comparison with the starting material. The sponge like behaviour of the material is a consequence of the increase in surface area. The novelty and the non obviousness of the present invention lies in the use of 98% pure soluble collagen developed by a novel process in our laboratory and also the use of surfactants in the preparation of the reconstituted collagen sponge. The use of surfactants facilitate the sponge to possess a large pore structure, that can be permissible for entrapping pharmaceutically active components. The sponge thus obtained can be used for entrapment of different drugs which facilitate the controlled release of the entrapped drugs. Effective cross-linking incorporated during the sponge preparation helps avoiding degradation of the final sponge. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention. EXAMPLE-I Hundred grams of foetal calf skin collected from a slaughter house, was thoroughly washed in plain water to free it from extraneous materials like the surrounding tissues. The washed stock was then chopped into small pieces of 2cm3 and the cut pieces were minced in Hobart Meat grinder. The temperature was maintained at 15° C by mixing crushed ice cubes along with the tendon pieces. The minced mass was then taken in a bath containing the scouring solution, which was prepared by dissolving 200 mg of Ethylene-oxide condensate of nonyl phenol in 300 ml water, with vigorous stirring. The temperature of the bath was maintained at 37°C and the stirring was continued for 8 hrs. Two hundred milligrams of sodium peroxide was dissolved in 300 ml of water taken in a beaker and the pH of the solution was adjusted to 10 for preparing a sliming solution. The scoured stock was then added to this sliming solution and stirring was continued for 4 hrs at a temperature of 30° C. The stock was then washed thoroughly with three changes of plain water to remove all the loose non - collagenous particles. Five grams of crystalline pepsin was added to 200 ml of water taken in a beaker at 6° C with constant stirring. The washed slimed stock was then put into the above enzyme bath with vigorous stirring. The pH of the bath was adjusted to 2 by adding H Cl. After a period of 48 hrs, the enzyme treated mass was fed into a polytron homogenisor maintained at 37° C and the homogenisation was done at 2500 rpm for 10 minutes till a viscous solution of collagenous tissue was obtained. The homogenate, thus formed, was taken in a beaker and was diluted with 100 ml of distilled water. 5 g of sodium chloride was then added to the beaker with stirring. When a precipitate of collagen tissue was formed at the bottom of the beaker, the precipitated collagen was centrifuged at 4° C and 10000 rpm and the sediment was then resolubilised in 500 ml of acetic acid at pH 2, while continuously stirring the solution for 90 min, till a clear viscous solution of collagen was obtained. The homogenous solution, thus obtained, was dialysed against 5 lit of 0.02 M di sodium hydrogen phosphate solution. The dialysate was centrifuged at 10000 rpm and the precipitate was redissolved in 500 ml of 0.5M acetic acid and dialysed against 51ts of distilled water for 10 hours. Five milligrams of Ethylene oxide condensate of nonyl phenol, 0.5 ml hexamethylene di isocyanate and 3 ml of antibiotic solution was added to the above collagen solution with vigorous for 60 min, till visibly very good froth was produced. This frothing solution was then taken in a container to ensure that the resulting sponge assumed the shape and size of it and finally the container was kept in liquid nitrogen till the contents were frozen. The frozen mass was lyophilised at -60° C and 80 Torr to get the reconstituted collagen sponge. The lyophilised sponge was properly packed in a polythene sachet which was hermetically sealed. The sachet was exposed to gamma ray irradiation from cobalt ° source for 30 sec to sterilise the dry sponge. EXAMPLE-II Hundred grams of Achilles tendon of a freshly slaughtered cow, collected from a slaughter house, was thoroughly washed in plain water to free it from extraneous materials like the surrounding tissues. The washed stock was then chopped into small pieces of 2cm3 and the cut pieces were minced in Hobart Meat grinder. The temperature was maintained at 15° C by mixing crushed ice cubes along with the tendon pieces. The minced mass was then taken in a bath containing the scouring solution, which was prepared by dissolving 300 mg of Sodium laurel sulphate in 300 ml water, with vigorous stirring. The temperature of the bath was maintained at 40°C and the stirring was continued for 3 hrs. Two hundred milligrams of potassium peroxide was dissolved in 300 ml of water taken in a beaker and the pH of the solution was adjusted to 10 for preparing a sliming solution. The scoured stock was then added to this sliming solution and stirring was continued for 4 hrs at a temperature of 40 ° C. The stock was then washed thoroughly with three changes of plain water to remove all the loose non - collagenous particles. Two grams of crystalline pepsin was added to 200 ml of water taken in a beaker at 4° C with constant stirring. The washed slimed stock was then put into the above enzyme bath with vigorous stirring. The pH of the bath was adjusted to 2.5 by adding HC1. After a period of 12 hrs, the enzyme treated mass was fed into a polytron homogenisor maintained at 30°C and the homogenisation was done at 2500 rpm for 10 minutes till a viscous solution of collagenous tissue was obtained. The homogenate, thus formed, was taken in a beaker and was diluted with 200 ml of distilled water. 15 g of potassium chloride was then added to the beaker with stirring. When a precipitate of collagen tissue was formed at the bottom of the beaker, the precipitated collagen was centrifuged at 6°C and 5000 rpm and the sediment was then resolubilised in 500 ml of acetic acid at pH 3, while continuously stirring the solution for 90 min, till a clear viscous solution of collagen was obtained. The homogenous solution, thus obtained, was dialysed against 5 lit of 0.02 M disodium hydrogen phosphate solution. The dialysate was centrifuged at 10000 rpm and the precipitate was redissolved in 500 ml of 0.5 M acetic acid and dialysed against 5 Its of distilled water for 24 hours. Two milligrams of sodium lauryl sulfate, 0.3 ml hexamethylene di isocyanate and 4 ml of antitumor drug solution were added to the above collagen solution with vigorous for 60 min, till visibly very good froth was produced. This frothing solution was then taken in a container to ensure that the resulting sponge assumed the shape and size of it and finally the container was kept in liquid nitrogen till the contents were frozen. The frozen mass was lyophilised at -60 ° C and 80 Torr to get the reconstituted collagen sponge. The lyophilised sponge was properly packed in a polythene sachet which was hermetically sealed. The sachet was exposed to gamma ray irradiation from cobalt 60 source for 30 sec to sterilise the dry sponge. EXAMPLE-m Hundred grams of bovine skin, preserved at -10 °C for a period of 7 days by adding 10 gms of common salt, was thoroughly washed in plain water to free it from extraneous materials like the surrounding ligament tissues. The washed stock was then chopped into small pieces of 2 cm3 and the cut pieces were minced in Hobart Meat grinder. The temperature was maintained at 20 ° C by mixing crushed ice cubes along with the tendon pieces. The minced mass was then taken in a bath containing the scouring solution, which was prepared by dissolving 350 mg of sodium laurel sulphate in 300 ml water, with vigorous stirring. The temperature of the bath was maintained at 35°C and the stirring was continued for 8 hrs. Three hundred and fifty milligrams of sodium meta bi sulphate was dissolved in 300 ml of water taken in a beaker and the pH of the solution was adjusted to 10 for preparing a sliming solution. The scoured stock was then added to this sliming solution and stirring was continued for 4 hrs at a temperature of 40 ° C. The stock was then washed thoroughly with three changes of plain water to remove all the loose non - collagenous particles. One gram of crystalline pepsin was added to 200 ml of water taken in a beaker at 2° C with constant stirring. The washed slimed stock was then put into the above enzyme bath with vigorous stirring. The pH of the bath was adjusted to 3 by adding HC1. After a period of 36 hrs, the enzyme treated mass was fed into a polytron homogenisor maintained at 35° C and the homogenisation was done at 2500 rpm for 10 minutes till a viscous solution of collagenous tissue was obtained. The homogenate, thus formed, was taken in a beaker and was diluted with 300 ml of distilled water. 5 g of sodium carbonate was then added to the beaker with stirring. When a precipitate of collagen tissue was formed at the bottom of the beaker, the precipitated collagen was centrifuged at 4 ° C and lOOOOrpm and the sediment was then resolubilised in 500 ml of formic acid at pH 2, while continuously stirring the solution for 90 min, till a clear viscous solution of collagen was obtained. The homogenous solution, thus obtained, was dialysed against 5 lit of 0.02 M disodium hydrogen phosphate solution. The dialysate was centrifuged at 10000 rpm and the precipitate was redissolved in 500 ml of 0.5M acetic acid and dialysed against 51ts of distilled water for 36 hours. Three milligrams of Iso octyl phenoxy poly ethoxy ethanol, 0.4 ml glutaraldehyde and 5 ml of growth regulator solution were added to the above collagen solution with vigorous for 60 min, till visibly very good froth was produced. This frothing solution was then taken in a container to ensure that the resulting sponge assumed the shape and size of it and finally the container was kept in liquid nitrogen till the contents were frozen. The frozen mass was lyophilised at -60 °C and 80 Torr to get the reconstituted collagen sponge. The sponge was folded and packed wet by placing into a glass tube containing 2 ml of a preserving fluid containing 95% v/v, Iso-propanol, 0.6% v/v, Ethylene oxide and 4.4% v/v, Water. The tube was finally hermetically sealed. The sealed tube was again packed in a sachet and sterilised in an ethylene oxide chamber. EXAMPLE-IV Hundred grams of foetal calf skin of a freshly slaughtered animal collected from a slaughter house, was thoroughly washed in plain water to free it from extraneous materials like the surrounding tissues. The washed stock was then chopped into small pieces of 2cm3 and the cut pieces were minced in Hobart Meat grinder. The temperature was maintained at 15° C by mixing crushed ice cubes along with the tendon pieces. The minced mass was then taken in a bath containing the scouring solution, which was prepared by dissolving 200 mg of Ethylene-oxide condensate of nonyl phenol in 300 ml water, with vigorous stirring. The temperature of the bath was maintained at 40°C and the stirring was continued for 8 hrs. Two hundred fifty milligrams of hydrogen peroxide was dissolved in 300 ml of water taken in a beaker and the pH of the solution was adjusted to 10 for preparing a sliming solution. The scoured stock was then added to this sliming solution and stirring was continued for 4 hrs at a temperature of 35 ° C. The stock was then washed thoroughly with three changes of plain water to remove all the loose non - collagenous particles. Two grams of crystalline trypsin was added to 200 ml of water taken in a beaker at 8° C with constant stirring. The washed slimed stock was then put into the above enzyme bath with vigorous stirring. The pH of the bath was adjusted to 3.5 by adding H Cl. After a period of 40 hrs, the enzyme treated mass was fed into a polytron homogenisor maintained at 30° C and the homogenisation was done at 2500 rpm for 10 minutes till a viscous solution of collagenous tissue was obtained. The homogenate, thus formed, was taken in a beaker and was diluted with 400 ml of distilled water. 5 g of sodium chloride was then added to the beaker with stirring. When a precipitate of collagen tissue was formed at the bottom of the beaker, the precipitated collagen was centrifuged at 8 ° C and 20000 rpm and the sediment was then resolubilised in 500 ml of formic acid at pH 4, while continuously stirring the solution for 90 min, till a clear viscous solution of collagen was obtained. The homogenous solution, thus obtained, was dialysed against 5 lit of 0.02 M di sodium hydrogen phosphate solution. The dialysate was centrifuged at 10000 rpm and the precipitate was redissolved in 500 ml of 0.5M acetic acid and dialysed against 51ts of distilled water for 48 hours. Five milligrams of sodium lauryl sulfate, 0.25 ml glutaraldehyde and 5 ml of antitumor drug solution was added to the above collagen solution with vigorous for 60 min, till visibly very good froth was produced. This frothing solution was then taken in a container to ensure that the resulting sponge assumed the shape and size of it and finally the container was kept in liquid nitrogen till the contents were frozen. The frozen mass was lyophilised at -60° C and 80 Torr to get the reconstituted collagen sponge. The sponge was folded and packed wet by placing into a glass tube containing 2 ml of a preserving fluid containing 95% v/v, Iso-propanol, 0.6% v/v, Ethylene oxide and 4.4% v/v, Water. The tube was finally hermetically sealed. The sealed tube was again packed in a sachet and sterilised in an ethylene oxide chamber. EXAMPLE- V Hundred grams of Achilles tendon collected from a slaughter house, was preserved at -20 °C for a period of 7 days by adding 10 gms of common salt, was thoroughly washed in plain water to free it from extraneous materials like the surrounding ligament tissues was thoroughly washed in plain water to free it from extraneous materials like the surrounding ligament tissues and superficial flexor tendon. The washed stock was then chopped into small pieces of 2cm3 and the cut pieces were minced in Hobart Meat grinder. The temperature was maintained at 10 ° C by mixing crushed ice cubes along with the tendon pieces. The minced mass was then taken in a bath containing the scouring solution, which was prepared by dissolving 200 mg of Ethylene-oxide condensate of nonyl phenol in 300 ml water, with vigorous stirring. The temperature of the bath was maintained at 37° C and the stirring was continued for 8 hrs. Three hundred milligrams .of sodium peroxide was dissolved in 300 ml of water taken in a beaker and the pH of the solution was adjusted to 10 for preparing a sliming solution. The scoured stock was then added to this sliming solution and stirring was continued for 4 hrs at a temperature of 30 ° C. The stock was then washed thoroughly with three changes of plain water to remove all the loose non - collagenous particles. One gram of crystalline papain was added to 200 ml of water taken in a beaker at 4° C with constant stirring. The washed slimed stock was then put into the above enzyme bath with vigorous stirring. The pH of the bath was adjusted to 3.5 by adding H Cl. After a period of 48 hrs, the enzyme treated mass was fed into a polytron homogenisor maintained at 37° C and the homogenisation was done at 2500 rpm for 10 minutes till a viscous solution of collagenous tissue was obtained. The homogenate, thus formed, was taken in a beaker and was diluted with 100 ml of distilled water. 10 g of sodium chloride was then added to the beaker with stirring. When a precipitate of collagen tissue was formed at the bottom of the beaker, the precipitated collagen was centrifuged at 6° C and 15000 rpm and the sediment was then resolubilised in 500 ml of hydrochloric acid at pH 3.5, while continuously stirring the solution for 90 min, till a clear viscous solution of collagen was obtained. The homogenous solution, thus obtained, was dialysed against 5 lit of 0.02 M di sodium hydrogen phosphate solution. The dialysate was centrifuged at 10000 rpm and the precipitate was redissolved in 500 ml of 0.5M acetic acid and dialysed against 51ts of distilled water for 48 hours. Five milligrams of Ethylene oxide condensate of nonyl phenol, 0.5 ml hexamethylene di isocyanate and 5 ml of antibiotic solution was added to the above collagen solution with vigorous for 60 min, till visibly very good froth was produced. This frothing solution was then taken in a container to ensure that the resulting sponge assumed the shape and size of it and finally the container was kept in liquid nitrogen till the contents were frozen. The frozen mass was lyophilised at -60° C and 80 Torr to get the reconstituted collagen sponge. The lyophilised sponge was properly packed in a polythene sachet which was hermetically sealed. The sachet was exposed to gamma ray irradiation from cobalt60 source for 30 sec to sterilise the dry sponge. The main advantages of the present invention are the following. 1. The present invention provides a process to prepare the collagen solution in purer form compared to of is able to solubilise much more collagenous tissues compared to that by the hitherto known processes in all the collagenous sources, including bovine skin and achilles tendons. 2. Pure soluble collagen, produced by this process, has minimal antigenicity and almost non immunogenic. It is very much compatible to human living tissues. 3. The product of this process is an economically viable method to prepare medical grade sponge. 4. Microscopically the reconstituted collagen sponge possesses a large pore structure which can be permissible for entrapping antibiotics. 5. The reconstituted collagen sponge can be used for entrapment of different drugs which facilitate the controlled release of the entrapped drugs 6. Effective cross-linking incorporated in the process helps avoiding degradation of the final sponge. 7. The additional advantage of this process is the production of soluble collagen an intermediate in production of reconstituted collagen sponges. 8. The soluble collagen, which is obtained as an intermediate product while making the sponge by the present process, has by itself a very high demand as a component in skin care products like creams, shampoos etc. in the cosmetic and pharmaceutical industry. 9. This new process has a great economical significance in a sense that it can open up new markets for both the reconstituted collagen sponge as well as the intermediate pure soluble collagen both in India and abroad. 10. The process suggests a method to utilise economically the slaughterhouse by- products like achilles tendon, foetal calf skin which are otherwise thrown away, thus making wealth out of waste. We Claim: 1. A process for the preparation of reconstituted collagen sponge which comprises i) adding the collagen free from non-collagenous materials to 0.2 to 5-1% w/v of foaming agent selected from surfactants, 0.5 - 1% v/v cross linking agent such as herein described and 1 - 5% v/v pharmaceutically and pharmacologically active component selected from antibiotics, biocides, anti tumor drugs, growth regulators ii) lyophilizing the resulting mixture, as formed in step (i), by conventional method, iii) sterilizing the lyophilized product, as formed in step (ii), by conventional method to obtain reconstituted collagen sponge. 2. A process, as claimed in claim 1, wherein the surfactants used is selected from Ethylene-oxide condensate of nonyl phenol, Sodium lauryl sulfate, Isooctyl phenoxy polyethoxyetthanol. 3. A process, as claimed in claims 1 and 2, wherein the amount of the foaming agent is preferably in the range of 0.5 - 0.75% by weight on the volume of the collagen solution. 4. A process, as claimed in claims 1 to 3, wherein the cross linking agents used are selected from Glutaraldehyde, Hexamethylene di isocyanate. 5. A process, as claimed in claims 1 to 5, wherein the method used for sterilization of the reconstituted collagen sponge is gamma ray irradiation, ethylene oxide treatment. 5. A process for the preparation of reconstituted collagen sponge, substantially as herein described with reference to the examples.

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