Title of Invention

A process for the preparation of a biopolymer scaffold for medical applications

Abstract The invention relates to a scaffold to aid in the tissue regeneration of tissue, such as bone, cartilage or soft tissue and as a container for delivery of bioactive molecules like growth factors. The scaffold is formed by the combination of the soluble biopolymer with a glycol, alkaline earth metal chloride and a cross linker. It comprises a polymer network having active agents, such as wound healing agents, incorporated therein. The compositions are suitable for use as or in wound dressings as it comprises methods and compositions for treating wounds. The matrix may be formed into any desired shape for treatment of wounds. The composition comprises a single density foam, either with or without cell seeding, which is not freeze dried, can be used to build living tissue equivalents or model tissue systems.
Full Text The present invention relates to a process for the preparation of a biopolymer scaffold for medical applications. More particularly, the present invention relates to a process for the preparation of biopolymeric scaffold, which is three dimensional in nature. The scaffold is envisaged to have enormous medical applications in respect of drug delivery, wound dressing and tissue-regenerating purpose. It also finds application as a container for delivery of bioactive molecules like growth factors and osteotropic factors. The structure of the scaffold is of paramount importance because of the fact that when implanted in the body as temporary structures, it provides a template that allows the body's own cells to grow and form new tissues while the scaffold is gradually absorbed.
Scaffolds are polymeric hollow fiber membranes, conventionally used for favouring tissue growth while treating bone defects. While naturally derived or synthetic materials may be fashioned into scaffolds, biocompatibility of the product poses a challenge for the medical application. Successful regeneration of cells can be achieved only with tissue-engineered cartilage implant if the seeded cells reveal an appropriate proliferation rate in the biodegradable scaffold together with the production of a new cartilage-specific extracellular matrix. Continuous efforts are therefore being made by the researchers to provide appropriate scaffolds exhibiting excellent biocompatibility.
Temenoff and Mikos (Biomaterials, 21,431-40,2000) and Lawrence and Vacanti (Journal of cellular Biochemistry Supplements, 30, 297-303,1998) have reported that the emerging field of tissue engineering may help to resolve many of the problems with regard to lack of donor organs or efficient organ substitutes. Tissue engineering involves the use of cells to regenerate the damaged tissue,
leaving only natural substances to restore organ function. It has been found that in order for the cells to maintain their tissue-specific functions once implanted, a substrate material must be inserted to aid in organization of the cells in three dimensions. In considering substrate materials, it is imperative to choose one that exhibits good biocompatibility. This means that the material must not elicit an unresolved inflammatory response nor demonstrate extreme immunogenicity or cytotoxicity. In addition, the mechanical properties of the scaffold must be sufficient so that it does not collapse during the patient's normal activities. As with all materials in contact with the human body, these scaffolds must be easily sterilizable to prevent infection.
According to Hutmacher (Biomaterials, 21, 2529-43, 2000), a scaffold should have the following characteristics: (i) three-dimensional and highly porous with an interconnected pore network for cell growth and flow transport of nutrients and metabolic waste; (ii) biocompatible and bio resorbable with a controllable degradation and resorption rate to match cell/tissue growth in vitro and/or in vivo; (iii) suitable surface chemistry for cell attachment, proliferation, and differentiation and (iv) mechanical properties to match those of the tissues at the site of implantation
Synthetic polymeric materials have been explored for use as tissue engineering scaffolds and drug delivery devices. Many investigators have concentrated on synthetic biodegradable polymers that are already approved by the Food and Drug Administration, USA, as reported by Yoon et al, (Biomaterials, 24, 2323-29, 2003), Hou et al, (Biomaterials, 24, 1937-47, 2003) and Jang et al (Journal of controlled release, 86, 156-68,2003). These are the poly (hydroxy esters) that are degraded by hydrolysis to products which can be metabolized and excreted.
Temenoff and Mikos (Biomaterials, 21, 431-40, 2000) and Schnabelrauch et al (Biomol Eng, 19, 295-8, 2002), have reported the synthesis, sterilization, toxicity, biocompatibility and clinical applications of scaffolds prepared with polylactic acid (PLLA) / polyglycolic acid (PGA) copolymers. These polymers offer distinct advantages in that their sterilizability and relative biocompatibility have been well documented. In addition, their degradation rates can be tailored to match that of new tissue formation. PLLA is more hydrophobic and less crystalline than PGA and degrades at a slower rate.
Mikos and Temenoff (Electronic Journal of Biotechnology, 3,114-19, 2000) have reported that in addition to degradation rate, certain physical characteristics of the scaffolds must be considered when designing a substrate to be used in tissue engineering. In order to promote tissue growth, the scaffold must have a large surface area to allow cell attachment. This is usually prepared by creating highly porous polymer foam. In these foams, the pore size should be large enough so that cells penetrate the pores, and the pores must be interconnected to facilitate nutrient and waste exchange by cells deep within the construct. These characteristics (porosity and pore size) are often dependent on the method of scaffold fabrication.
According to Angela et al (Biomaterials, 24, 481-89, 2003) the biodegradable porous polymer scaffolds are widely used in tissue engineering to provide a structural template for cell seeding and extracellular matrix formation. Scaffolds must often possess sufficient structural integrity to temporarily withstand functional loading in vivo or cell traction forces in vitro. Poly (lactic acid) (PLA) and poly (L-lactic-co-glycolic acid) (PLGA), poly (Llactide-co-DL-lactide (PLDL) have been widely used as three-dimensional (3D) polymer scaffolds for cell transplantation and drug delivery applications, since they are biodegradable and biocompatible. It is important that the
scaffolds are highly porous enough to allow a high density of cells to be seeded, and when the cell-device construct is implanted in the body, they should permit the facile invasion of blood vessels for the supply of nutrients to the transplanted cells. To fulfill these requirements, the polymer scaffolds should have several physical characteristics such as high porosity, a large surface area, a large pore size, and a uniformly distributed and highly interconnected pore structure throughout the matrix.
Leong et al. (U.S. Pat. No. 5,686,091) disclosed a method in which biodegradable porous polymer scaffolds are prepared by molding a solvent solution of the polymer under conditions permitting spinodal decomposition, followed by quenching of the polymer solution in the mold and sublimation of the solvent from the solution. A uniform pore distribution is disclosed. A biomodal pore distribution would increase the degree of pore interconnectivity by creating additional channels between the pores, thereby increasing total porosity and surface area
Healy et al. (U.S. Pat. No. 5,723,508) have disclosed a method in which biodegradable porous polymer scaffolds are prepared by forming an emulsion of the polymer, a first solvent in which the polymer is soluble, and a second polymer that is immiscible with the first solvent, and then freeze-drying the emulsion under conditions that do not break the emulsion or throw the polymer out of solution. This process, however, also produces a more uniform pore size distribution, with the majority of the pores ranging from 9 to 35 microns in diameter.
However, these synthetic polymers possess a surface chemistry that does not promote cell adhesion. In addition, they produce a high local concentration of acidic by-products during degradation that can induce an adverse inflammatory response or create a local environment in the scaffold that may not favor the biological activity of cells being cultured for tissue engineering purposes. Furthermore, these processing
techniques (excluding fibre meshes) are dependent on a randomly distributed pore generator to form the pores within the scaffold. Consequently, these processing techniques are unable to precisely control pore size, ensure reliable interconnection and control the distribution of pores within the scaffold. In addition, these processing techniques cannot produce a controlled internal architecture, which would favor the growth of blood vessels.
The limitations associated with the synthetic polymers have prompted the researchers to explore possibilities of using biopolymers. A biopolymer is a naturally occurring polymeric substance formed from individual molecules in a biological system or organism. Biopolymers can also be man-made by manipulation of the individual molecules once obtained outside the biological system or organism. The biopolymer is suitable for introduction into a living organism, e.g., a mammal, e.g., a human. The biopolymer is non-toxic and bio absorbable when introduced into a living organism and any degradation products of the biopolymer should also be non-toxic to the organism. The biopolymers of the invention can be formed into biocompatible forms, e.g., biocompatible scaffolds, biocompatible foams, biocompatible gels, biocompatible constructs which include biocompatible fibers, e.g., collagen fibers, biocompatible fabrics, e.g., collagen fabrics, all with or without other extra cellular matrix macromolecules. Examples of molecules which can form biopolymers and which can be used in the present invention include collagen, laminin, elastin, fibronectin, fibrinogen, gelatin, and polysaccharides. A combination or mixture of one or more biopolymers can be used to form the biocompatible forms. A preferred molecule for biopolymer scaffold production is collagen.
Biopolymers, such as animal proteins and plant polysaccharides, have been used in recent years in a number of diverse applications, including biomedical
applications. According to Friess (European journal of pharmaceutics and bio pharmaceutics 45,113-36,1998) the use of collagen as a biomaterial is currently undergoing regeneration in the field of tissue engineering. Rocha et al (Biomaterials, 23,449-56,2002) and Fujioka et al (Advanced drug delivery Reviews, 31, 247-66,1998) have reported the use of collagen, the major protein of skin and connective tissue, in wound dressings, as well as surgical sponges. Lee et al (International Journal of Pharmaceutics, 221, 1-22, 2001) have reported the use of collagen for tissue engineering including skin replacement, bone substitutes, and artificial blood vessels and valves. They also have reported that open porous structure of the collagen sponge has a great potential for the delivery of biologically active agents such as growth factors, drugs, cytokines, etc. and other therapeutic agents. The purified collagen material can be engineered into a variety of tissue matrices for biomedical applications. These tissue matrices can be in the porous, tubular, membrane, filament and fibrillar forms. Roche et al (Biomaterials, 22, 9 -18, 2001) have reported the use of cross linked collagen sponges seeded with fetal bovine chondrocytes for cartilage tissue engineering.
Collagen has been identified as an excellent biomaterial for medical application primarily because of its inherent compatibility with human body. Ries et al (U.S. Pat. No. 4,066,083) have disclosed the procedure for the preparation of collagen solution from pigskin. The skin is finely divided, degreased using detergent, washed, and digested with pepsin to give a viscous suspension, and the collagen precipitated by addition of saturated salt solution. The precipitate is suspended in acid, re precipitated as a fibrous white precipitate in salt solution, washed as many times as desired, and desalted by washing with alcohol. The purified collagen is then
suspended in acid solution and freeze-dried. It is sterilized by gamma-irradiation, which may degrade or cross link the preparation.
Ho et al (Journal of controlled release, 77, 2001, 97-105) have reported the preparation of telopeptide poor collagen from porcine skin. The collagen was obtained by acetone treatment to remove fat, followed by homogenisation and digestion with pepsin at pH 2.0 for a desired period. The pH of the supernatant was adjusted to 10.0 with sodium hydroxide to inactivate and then it was adjusted to pH 7.0 with H Cl. The precipitated collagen was then dissolved in a 3% acetic acid solution. The collagen thus obtained was used for the preparation of collagen gels.
Depending on the characteristics, it may be of different types, which may range from Type-I to Type-XIV. A preferred combination of collagen types includes collagen type I, collagen type III, and collagen type IV. Preferred mammalian tissues for extraction of pure collagenous substance in respect of making biopolymer for medical applications include entire mammalian fetuses, e.g., porcine fetuses, dermis, tendon, muscle and connective tissue. As a source of collagen, fetal tissues are advantageous because the collagen in the fetal tissues is not as heavily crosslinked as in adult tissues. Thus, when the collagen is extracted using acid extraction, a greater percentage of intact collagen molecules is obtained from fetal tissues in comparison to adult tissues. Fetal tissues also include various molecular factors, which are present in normal tissue at different stages of animal development
Roberts et al (Advances in Drug Delivery Reviews, 54: 459-76, 2002) reported that Polyethylene glycol (PEG) is a highly investigated polymer for the covalent modification of biological macromolecules and surfaces for many pharmaceutical and biotechnological applications. In the modification of biological macromolecules, peptides and proteins are of extreme importance. Reasons for
PEGylation (i.e. the covalent attachment of PEG) of peptides and proteins are numerous and include shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake by the reticuloendothelial system (RES), and preventing recognition and degradation by proteolytic enzymes. PEG conjugation also increases the apparent size of the polypeptide, thus reducing the renal filtration and altering bio distribution. An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to the peptide or protein.
According to Sachols (Biomaterials, 24, 1487-97, 2003) collagen may be the ideal scaffold material as it is the major component of the extra cellular matrix. Therefore, it has a more native surface, relative to synthetic polymers, which favors cellular attachment as well as being chemotactic to cells and. Furthermore, collagen substrates can modify the morphology, migration and in certain cases the differentiation of cells. These properties of collagen emphasize its significance in tissue regeneration and its value as a scaffold material. However, to date, collagen scaffolds have been fabricated into foam structures via the freeze-drying and critical point drying techniques. Therefore, the diffusion constraints arising from the scaffold foam design does not allow cell migration deep into to the scaffold.
There are a very few scaffolds reported in the literature involving collagen. Silver et al (U.S. Pat. No. 4,970,298) have reported a biodegradable collagen matrix exhibiting application as a wound implant. The matrix is formed by freeze drying an aqueous dispersion containing collagen, cross-linking the collagen via two cross-linking steps and freeze-drying the cross-linked matrix. The matrix may also contain hyaluronic acid and fibronectin.
Liu, et al (US patent, 5,972,385) have reported a Collagen-polysaccharide matrix for bone and cartilage repair where in the matrix and a method for preparing it
are provided to support the growth of tissue, such as bone, cartilage or soft tissue. A polysaccharide is reacted with an oxidizing agent to open sugar rings on the polysaccharide to form aldehyde groups. The aldehyde groups are reacted to form covalent linkages to collagen.
Li et al (US Patent, 5,206,028) have reported the preparation of dense collagen membrane matrices for medical uses where in the invention was directed to collagen membranes having physical and biological properties, which make them suitable and desirable for medical uses, particularly as a periodontal barrier. The membranes are characterized by having surface roughness morphology similar to leather and are translucent. Moreover, these membranes do not swell appreciably upon being wetted so as to maintain their overall bulk density.
The above bio absorbable scaffolds implants were prepared by suspending the material in a solvent followed by either freeze-drying or solvent drying. However, the major limitation associated with these processes is that it is generally difficult to control the pore size and density of sponge materials made in this way. Moreover, the resorption of these scaffolds is slowed drastically by chemical cross-linking of the biopolymer. The resulting bio polymeric scaffold is liable to be associated with some immune reactions, while using as implant, as well as do not hold the entrapped bioactive molecules for long periods. Another limitation is that these scaffolds are principally prepared by lyophilizing or freeze drying, which is not a cost-effective method of manufacturing. Alternatively, freeze-drying the construct can kill the cells of the scaffolds. Freeze drying eliminates living material, but leaves the deposited proteins in their natural states.
he main object of this present invention is to provide a process for the preparation of a biopolymer scaffold for medical applications, which obviates the limitations as stated above.
Another object of the present invention is to provide a process to ensure that the biopolymer molecule is stabilized during the process
Yet another object of the present invention is to provide a biopolymeric scaffold that is three-dimensional in nature.
Still another object of the present invention is to provide a process to ensure that the preparation of the scaffold is highly cost effective method by avoiding the freeze-drying.
Yet another object of the present invention is to provide a process for the preparation of biopolymeric scaffold with porosity in the range of 50 to 500 fjrn
Still another object of the present invention is to provide a process to create scaffolds of any form or shape, e.g., strips, sheets, tubes, etc
Accordingly the present invention provides a process for the preparation of a biopolymer scaffold for medical applications, which comprises
i) treating biopolymer solution selected from collagen of any individual type or different types in any combination, laminin, elastin, fibronectin, fibrinogen, gelatin, polysaccharides, poly-1-amino acids, either individually or in any combination having concentration in the range of 5-10%w/v, with not less than 1% w/v of glycol and 0.02 - 0.2%w/v of alkaline earth metal chloride, optionally in the presence of not more than 5% of macromolecules selected from growth factors such as herein described, extracellular matrix proteins, proteoglycans, glycosaminoglycans polysaccharides and living cells such as herein described, either individually or in any combination, at a
37°C followed by homogenization of the resulting solution by known method at a speed of not less than 6500 rpm for a period of 1-5 minutes at a temperature not exceeding 37°C to form a viscous frothy mass,
ii) subjecting the frothy mass, as obtained in step(i), optionally to conventional cross linking such as ultraviolet treatment, or treatment with 0.2-1% v/v of conventional chemical cross linker, either individually or in combination or succession and optionally not more than 5% w/v of pharmacologically active compounds as herein described at a temperature not exceeding 40°C, preferably below 37°C, followed by homogenization of the resulting mass by known method at a speed of not less than 6500 rpm for a period of 1-5 minutes at a temperature not exceeding 37°C and subsequent casting of the resulting mass.,by known method in any shape followed by conventional drying at a temperature not exceeding 40°C and sterilization by known method to get biopolymer scaffold. In an embodiment of the present invention the biopolymer used may be
selected from collagen of any individual type or different types in any combination, laminin, elastin, flbronectin, flbrinogen, gelatin, polysaccharides, poly-1-amino acids, either individually or in any combination.
In another embodiment of the present invention the glycol used may be selected from ethylene glycol, propylene glycol, mono ethylene glycol, mono propylene glycol, di ethylene glycol, tri ethylene glycol, glycol mono ethyl ether, polyethylene glycol, polypropylene glycol.
In yet another embodiment of the present invention, the alkaline earth metal chloride used may be selected from magnesium chloride, calcium chloride, strontium chloride, barium chloride
In still another embodiment of the present invention, growth factor used may be selected from but not limited to, platelet derived growth factors (PDGF),
e.g., PDGF AA, PDGF BB; insulin-like growth factors (IGF), e.g., IGF-I, IGF-II; fibroblast growth factors (FGF), e.g., acidic FGF, basic FGF,. beta.-endothelial cell growth factor, FGF 4, FGF 5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF), e.g., TGF-P1, TGF-.beta.l.2, TGF-.beta.2, TGF-.beta.3, TGF-.beta.5; bone morphogenic proteins (BMP), e.g., BMP 1, BMP 2, BMP 3, BMP 4; vascular endothelial growth factors (VEGF), e.g., VEGF, placenta growth factor; epidermal growth factors (EGF), e.g., EGF, amphiregulin, betacellulin, heparin binding EGF; interleukins, e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14; colony stimulating factors (CSF), e.g., CSF-G, CSF-GM, CSF-M; nerve growth factor (NGF); stem cell factor; hepatocyte growth factor, and ciliary neurotrophic factor, either individually or in any combination.
In yet another embodiment of the present invention, extracellular matrix proteins used may be selected from fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagens, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, kalinin, either individually or in any combination.
In still another embodiment of the present invention, proteoglycan used may be selected from dermatan sulfate proteoglycans, keratan sulfate proteoglycans, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, either individually or in any combination.
In yet another embodiment of the present invention, glycosaminoglycan used may be selected from heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, either individually or in any combination.
In yet another embodiment of the present invention, polysaccharide used may be selected from heparin, dextran sulfate, chitin, alginic acid, pectin, xylan, either individually or in any combination.
In still another embodiment of the present invention, living cells used may be selected from, but are not limited to, epithelial cells, e.g., keratinocytes, adipocytes, hepatocytes, neurons, glial cells, astrocytes, podocytes, mammary epithelial cells, islet cells; endothelial cells, e.g., aortic, capillary and vein endothelial cells; and mesenchymal cells, e.g., dermal fibroblasts, mesothelial cells, stem cells, osteoblasts, smooth muscle cells, striated muscle cells, ligament fibroblasts, tendon fibroblasts, chondrocytes, and fibroblasts, either individually or in any combination .
In yet another embodiment of the present invention, chemical crosslinker used may be selected from glutaraldehyde, formaldehyde, acrylamide; carbodiimides, such as l-ethyl-3-(dimethyl aminopropyl)carbodiimide; diones such as 2, 5-hexanedione; diimidates, such as dimethyl suberimidate; bisacrylamides, such as N, N'-methylenebisacrylamide.
In still another embodiment of the present invention, the pharmacologically active components used may be selected from antibiotics such as penicillin, cefalosporins, tetracyclines, streptomycin, gentamicin; sulfonamides; antifungal drugs such as myconazolle, anti-inflammatory agents are cortisone, a synthetic derivative thereof, or any synthetic anti-inflammatory drugs and growth factors such as fibroblast growth factor, platelet derived growth factors, transforming growth factors, insulin-like growth factors, etc; differentiating factors such as bone morphogenetic proteins, either individually or in any combination.
In still another embodiment of the present invention, the known method of sterilization used may be such as gamma ray irradiation, cobalt 6(), ethylene oxide treatment.
The process of the present invention is described below in detail.
The biopolymer solution is prepared by the following conventional method.
A source of biopolymer 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 tissue, at a temperature of max. 40° C. The scoured mass is then slimed with minimum 0. 2% of sliming agent at 30-40°C. The slimed mass is then washed thoroughly to make the same free from non-biopolymeric 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 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 biopolymer 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 biopolymer is dissolved in 200-400% v/v of an acid at a pH in the range of 2- 4. The homogenous solution, thus obtained, is dialysed against di sodium hydrogen phosphate solution. The dialysate is centrifuged at 10000- 20000 rpm and the precipitate is redissolved in 0.5M acetic acid and dialysed against distilled water for 10-48 hours to get pure biopolymer solution.
he biopolymer solution having concentration in the range of 5-10%w/v, as prepared by the above process, is treated with not less than 1 % w/v of glycol and 0.02 - 0.2%w/v of alkaline earth metal chloride, optionally in the presence of not more than 5% of macromolecules necessary for cell growth, morphogenesis, differentiation, and tissue building, that includes but not limited to growth factors, extracellular matrix proteins, proteoglycans, glycosaminoglycans, polysaccharides, living cells, either individually or in any combination, at a temperature in the range of 4-37°C. The resulting solution is homogenized by known method at a speed of not less than 6500 rpm for a period of 1-5 minutes at a temperature not exceeding 37°C to form a viscous frothy mass, which is treated with 0.2-1% v/v of conventional cross linker and optionally not more than 5% w/v of pharmacologically active compound at a temperature not exceeding 40°C, preferably below 37°C. The resulting solution is again subjected to homogenization by known method at a speed of not less than 6500rpm for a period of 1-5 minutes at a temperature not exceeding 37°C. The resulting mass is cast in any shape conventionally. It is then dried conventionally at a temperature not exceeding 40°C and subsequently sterilized by known method to get biopolymer scaffold.
The inventive step of the present invention lies in the treatment of the biopolymer solution with glycol along with alkaline earth metal chloride to get three-dimensional porous structure of the scaffold that is capable of obtaining a special texture, at a temperature as high as 40°C, unlike conventional scaffolds, which are prepared by freeze drying, thus providing a cost effective approach for making multipurpose biopolymer scaffold of wider varieties.
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-1
The biopolymer solution was obtained in the following manner. Hundred grams of fetal calfskin collected from a slaughterhouse, 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 8hrs.
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 HC1. 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 dialyzed 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 0.5 M acetic acid and dialyzed against Sits of distilled water for 10 hours to get pure collagen solution. The pure collagen solution was freeze - dried and stored at 4°C
Fifty millilitres of pure 5% collagen and gelatin solution was taken in a round bottom flask and 500mg of poly ethylene glycol, 200 mg of, magnesium chloride, was added and homogenized at 6500rpm for 1 minute at a temperature of 37oC. To the resulting mixture O.lml of l-ethyl-3 - (dimethyl aminopropyl)carbodiimide and SOOmg of streptomycin, were added with continuous shaking. The above mixture was then fed into an Ultratuurax homogenisor maintained at 10° C and the homogenisation was done at 6500rpm for 1 minutes till a viscous froth of collagen was obtained. The frothy mass was cast into a scaffold by casting the same in a Teflon coated tray and drying the same in a laminar flow hood in a sterile and dust free atmosphere to get the collagenous scaffold.
The dried scaffold was properly placed 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 scaffold. EXAMPLE-2
The biopolymer solution was obtained in the following manner. Hundred grams of Achilles tendon of a freshly slaughtered cow, collected from a slaughterhouse, was thoroughly washed in plain water to free it from extraneous
aterials 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 150 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 tor 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 to get pure collagen solution. The pure collagen solution was freeze-dried and stored at 4°C
Fifty millilitres of pure 6% collagen solution was taken in a round bottom flask and Ig of polypropylene glycol, 20 mg of calcium chloride, was added was then fed into an Ultratuurax homogenisor and homogenized at 7000 rpm for a period of 2 minute at 10°C temperature. To the resulting mixture 0.2ml glutaraldehyde and Ig of cortisone, were added with continuous shaking. The above mixture maintained at lOo C and the homogenisation was done at 7000 rpm for 2 minutes till a viscous frothy mass of collagen was obtained. The mixture was cast into a scaffold by casting the same in a Teflon coated tray and drying the same in a laminar flow hood in a sterile and dust free atmosphere to get the collagenous scaffold.
The dried scaffold was properly placed 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 scaffold. The lyophilised scaffold was properly placed 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 scaffold. EXAMPLE-3
The biopolymer solution was obtained in the following manner. 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 10000 rpm 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 dialyzed 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 to get pure collagen solution. The pure collagen solution was freeze-dried and stored at 4°C
Fifty millilitres of pure 7% fibronectin solution was taken in a round bottom flask and 1.5g of mono ethylene glycol, 80 mg of, strontium chloride, was added and homogenized at 7500 rpm for 3 minutes at a temperature of 37oC. To the resulting mixture 0.3ml of N, N'-methylenebisacrylamide and 1.5g of cefalosporins, were added with continuous shaking. The above mixture was then fed into an Ultratuurax homogenisor maintained at 10° C and the homogenisation was done at 7500 rpm for 3 minutes till a viscous froth of collagen was obtained. The frothy mass was cast into a scaffold by casting the same in a Teflon coated tray and drying the same in a laminar flow hood in a sterile and dust free atmosphere to get the collagenous scaffold.
The scaffold was folded and packed wet by placing into a glass tube containing 2 ml of 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-4
The biopolymer solution was obtained in the following manner. Hundred grams of porcine fetal skin of a freshly slaughtered animal collected from a slaughterhouse, 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 150C 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 HC1. 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 to get pure collagen solution. The pure collagen solution was freeze- dried and stored at 4°C
Fifty millilitres of pure 8% collagen solution was taken in a round bottom flask and 2.0g of mono ethylene glycol, 120 mg of, strontium chloride, was added and homogenized at 1000 rpm for 4 minutes at a temperature of 37oC. To the resulting mixture 0.4ml of hexamethylene di isocyanite and 0.2g of heparan sulfate, were added with continuous shaking. The above mixture was then fed into an Ultratuurax homogenisor maintained at 10° C and the homogenisation was done at 10000 rpm for 4 minutes till a viscous froth of collagen was obtained. The frothy mass was cast into a scaffold by casting the same in a Teflon coated tray and drying the same in a laminar flow hood in a sterile and dust free atmosphere to get the collagenous scaffold The air-dried scaffold was properly placed in a polythene sachet, which was hermetically sealed. The sachet was exposed to gamma ray irradiation from cobalt 60 source for 30 sec to sterilize the dry scaffold. EXAMPLE-5
The biopolymer solution was obtained in the following manner. Hundred grams of fetal porcine skin 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 to get pure collagen solution. The pure collagen solution was freeze-dried and stored at 4°C
Fifty millilitres of pure 9% collagen solution was taken in a round bottom flask and 3.0g of mono ethylene glycol, 160 mg of, strontium chloride, was added and homogenized at 12000 rpm for 5 minutes at a temperature of 37oC. To the resulting mixture 0.5ml of hexamethylene di isocyanite and 2.5g of streptomycin, were added with continuous shaking. The above mixture was then fed into an Ultratuurax homogenisor maintained at 10° C and the homogenisation was done at 12000 rpm for 5 minutes till a viscous froth of collagen was obtained. The frothy mass was cast into a scaffold by casting the same in a Teflon coated tray and drying the same in a laminar flow hood in a sterile and dust free atmosphere to get the collagenous scaffold.
The scaffold 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-6
The biopolymer solution was obtained in the following manner. Hundred grams of Achilles tendon of a freshly slaughtered cow, collected from a slaughterhouse, 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 150 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
300ml 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 Ultratuurax homogenisor maintained at 300C 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 It. of distilled water for 24 hours to get pure collagen solution. The pure collagen solution was freeze-dried and stored at 4°C
Fifty millilitres of pure 10% collagen solution was taken in a round bottom flask and 5.0g of mono ethylene glycol, 200 mg of, strontium chloride, was added and homogenized at 12000 rpm for 5 minutes at a temperature of 37oC. To the resulting mixture 0.5ml of hexamethylene di isocyanite and Twenty microliters of a growth factor solution (equivalent to 30 |lg of growth factor in deionized water), were added with continuous shaking. The above mixture was then fed into an Ultratuurax homogenisor maintained at 10° C and the homogenisation was done at 12000 rpm for 5 minutes till a viscous froth of collagen was obtained. The frothy mass was cast into a scaffold by casting the same in a Teflon coated tray and drying the same in a laminar flow hood in a sterile and dust free atmosphere to get the collagenous scaffold.
The scaffold was properly placed 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 scaffold.
The main advantages of the present invention are the following.
1. It is a simple method to prepare a biopolymer scaffold useful for drug delivery,
wound dressing and tissue regeneration applications,
2. The glycol used in the preparation of the scaffold stabilizes the porous structure
without deactivating the biological properties of collagen. These porous
composite materials could function as a scaffold to organize tissue in-growth.
3. The alkaline metal chloride salt ensures that the scaffold texture is otherwise soft
and also allows absorbing extra water, maintaining moisture and fastening the
drying process of the scaffold.
4. The scaffold can be placed at the tumor or disease site to deliver a drug at dose
levels considerably stronger than the body would normally tolerate with a
systemic delivery approach. After drug delivery, the open cell structure of the
implant remains for a period of time to promote regeneration of healthy tissues
5. The scaffold is highly porous, flexible and has better stability.
6. The scaffold is porous and allows faster tissue in growth.
7. The product of this process is an economically viable method to prepare medical
grade scaffold.
8. The biodegradable biopolymer scaffold can be used for entrapment of different
drugs which facilitate the controlled release of the entrapped drugs.
9. Effective cross-linking incorporated in the process helps avoiding degradation of
the final scaffold.
10. The method of preparing the scaffold if very cost effective.
11. The soluble collagen, which is obtained as an intermediate product while making
the scaffold by the present process, has by itself a very high demand as a
component in skin care products like creams, shampoo etc. in the cosmetic and
pharmaceutical industry.





WE CLAIM
1. A process for the preparation of a biopolymer scaffold for medical
applications, which comprises
i) treating biopolymer solution selected from collagen of any individual type or different types in any combination, laminin, elastin, fibronectin, fibrinogen, gelatin, polysaccharides, poly-1-amino acids, either individually or in any combination having concentration in the range of 5-10%w/v, with not less than 1% w/v of glycol and 0.02 - 0.2%w/v of alkaline earth metal chloride, optionally in the presence of not more than 5% of macromolecules selected from growth factors such as herein described, extracellular matrix proteins, proteoglycans, glycosaminoglycans polysaccharides and living cells such as herein described either individually or in any combination at a temperature in the range of 4-37°C followed by homogenization of the resulting solution by known method at a speed of not less than 6500 rpm for a period of 1-5 minutes at a temperature not exceeding 37°C to form a viscous frothy mass,
ii) subjecting the frothy mass, as obtained in step(i), optionally to conventional cross linking such as ultraviolet treatment, or treatment with 0.2-1% v/v of conventional chemical cross linker, either individually or in combination or succession and optionally not more than 5% w/v of pharmacologically active compounds as herein described at a temperature not exceeding 40°C, preferably below 37°C, followed by homogenization of the resulting mass by known method at a speed of not less than 6500 rpm for a period of 1-5 minutes at a temperature not exceeding 37°C and subsequent casting of the resulting mass by known method in any shape followed by conventional drying at a temperature not exceeding 40°C and sterilization by known method to get biopolymer scaffold.
2. A process, as claimed in Claim 1 , wherein the glycol used is selected from
ethylene glycol, propylene glycol, mono ethylene glycol, mono propylene
glycol, di ethylene glycol, tri ethylene glycol, glycol mono ethyl ether,
polyethylene glycol, polypropylene glycol.
3. A process, as claimed in Claims 1 and 2, wherein the alkaline earth metal
chloride used is selected from magnesium chloride, calcium chloride,
strontium chloride, barium chloride
4. A process, as claimed in Claims 1 to 3, wherein growth factor used is such as,
platelet derived growth factors (PDGF), PDGF, PDGF BB; insulin-like
growth factors IGF, IGF-I, IGF-I1; fibroblast growth factors (FGF), acidic
FGF, basic FGF,. beta.-endothelial cell growth factor, FGF 4, FGF 5, FGF 6,
FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF), TGF-PI, TGF-
beta,1.2, TGF-beta2, TGF-beta3, TGF-beta5; bone morphogenic proteins
0MPy, BMP 1, BMP 2, BMP 3, BMP 4; vascular endothelial growth factors
(VEGF), VEGF, placenta growth factor; epidermal growth factors (EGF),
EGF, amphiregulin, betacellulin, heparin binding EGF; interleukins, IL-1, IL-
2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14;
colony stimulating factors (CSF), CSF-G, CSF-GM, CSF-M; nerve growth
factor (NGF); stem cell factor; hepatocyte growth factor, and ciliary
neurotrophic factor, either individually or in any combination.
5. A process, as claimed in claims 1 to 4, wherein extracellular matrix proteins
used is selected from fibronectin, laminin, vitronectin, tenascin, entactin,
thrombospondin, elastin, gelatin, collagens, fibrillin, merosin, anchorin,
chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin,
epinectin, hyaluronectin, undulin, epiligrin, kalinin, either individually or in any combination.
6. A process, as claimed in claims 1 to 5, wherein proteoglycan used is selected
from dermatan sulfate proteoglycans, keratan sulfate proteoglycans,
chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, either
individually or in any combination.
7. A process, as claimed in claims 1 to 6, wherein glycosaminoglycan used is
selected from heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan
sulfate, hyaluronic acid, either individually or in any combination.
8. 9. A process, as claimed in claims 1 to 7, wherein polysaccharide used is
selected from heparin, dextran sulfate, chitin, alginic acid, pectin, xylan,
either individually or in any combination.
9. A process, as claimed in claims 1 to 8, wherein living cell used is selected
from epithelial cells- keratinocytes, adipocytes, hepatocytes, neurons, glial
cells- astrocytes, podocytes, mammary epithelial cells, islet cells; endothelial
cells- aortic, capillary and vein endothelial cells; and mesenchymal cells-
dermal fibroblasts, mesothelial cells, stem cells, osteoblasts, smooth muscle
cells, striated muscle cells, ligament fibroblasts, tendon fibroblasts,
chondrocytes, and fibroblasts, either individually or in any combination .
10. A process, as claimed in claims 1 to 9, wherein chemical crosslinker used is
selected from glutaraldehyde, formaldehyde, acrylamide; carbodiimides, such
as 1-ethy 1-3-(dimethyl aminopropyl)carbodiimide; diones such as 2,5-
hexanedione; diimidates, such as dimethyl suberimidate; bisacrylamides,
such as N,N'-methylenebisacrylamide.
11. A process, as claimed in claims 1 to 10, wherein the pharmacologically active
component used is selected from antibiotics such as penicillin, cefalosporins,
tetracyclines, streptomycin, gentamicin; sulfonamides; antifungal drugs such
as myconazolle, anti-inflammatory agents are cortisone, a synthetic derivative
thereof, or any synthetic anti-inflammatory drugs and growth factors such as
fibroblast growth factor, platelet derived growth factors, transforming growth
factors, insulin-like growth factors, differentiating factors such as bone
morphogenetic proteins, either individually or in any combination.
12. A process, as claimed in claims 1 to 11, wherein known method of
sterilization method used is selected from gamma ray irradiation, cobalt 60,
ethylene oxide treatment
13. A process for the preparation of a biopolymer scaffold for medical
applications substantially as herein described with reference to the examples.





Documents:

1054-del-2003-abstract.pdf

1054-del-2003-claims.pdf

1054-del-2003-complete specification (granted).pdf

1054-del-2003-correspondence-others.pdf

1054-del-2003-correspondence-po.pdf

1054-del-2003-description (complete).pdf

1054-del-2003-form-1.pdf

1054-del-2003-form-19.pdf

1054-del-2003-form-2.pdf

1054-del-2003-form-3.pdf


Patent Number 199822
Indian Patent Application Number 1054/DEL/2003
PG Journal Number 29/2008
Publication Date 26-Sep-2008
Grant Date 05-Jan-2007
Date of Filing 28-Aug-2003
Name of Patentee Council of Scientific and Industrial Research
Applicant Address Rafi Marg, New Delhi
Inventors:
# Inventor's Name Inventor's Address
1 Dasari Vijaya Ramesh Central Lether Research Institute, Chennai
2 Praveen Kumar Sehgal Central Lether Research Institute, Chennai
PCT International Classification Number A 61K 31/00, 128K
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA