Title of Invention

"A PROCESS FOR THE PREPARATION OF NOVEL BIO-INORGANIC COMPOSITE USEFUL FOR BONE SUBSTITUTION "

Abstract

A process for the preparation of novel bio-inorganic composite useful for bone substitution which comprises: :) washing cancellous rich bones by conventional manner using saline buffer and water successively, ii) degreasing the bones by defattening solvents such as herein described in multiples stages for 8-11 hrs , wherein amount of solvent is 100% w/v of the weight of washed bone, iii) conditioning the defatted bones with 4-8 times w/v aqueous organic solvent medium , such as herein described , over a period of 6-8 hrs., iv) removing proteoglycans by treating the conditioned bones, as formed in step(iii), with a dissociative extracting solution selected from urea, guanidium monochloride, calcium chloride in the range of 20-50 % aqueous solution of 200-400% w/w of the proteoglycan , over a period of 12-24 hrs. at pH in the range of 5.6-5.8 , at temperature ranging 4-200 C, v) demineralization of the bones using demineralizing agents such as herein described, at pH in the range of 1-5 over 48-96 hrs., vi) removing non/collagenous proteins as an optional step, using 7-12 times w/v of a conventional dissociative extracting solution as defined above over a period of 48-72 hrs at pH 7.2-7.4, vii) lyophilizing the collagen matrix by the conventional manner at -70 C, viii) treating the denatured collagen matrix, as prepared in step (vii), with saturated calcifying solution 1 - 3 % w/v dissolved in acidic water at a pH ranging 1 -2.5 , followed by adjusting at pH in the range of 4.5 - 8.5 to get phase transformed calcium phosphate crystals and incorporating to it an antibiotic as defined herein at a range 0.25 - 1.25 % v/v , and keeping for 4-8 days at 30-37°C, ix) lyophilizing the resulting composite, as formed in step (viii) followed by sterilization by conventional way to get desired bio - inorganic composite.

Full Text

The present invention relates to a process for the preparation of novel bio-inorganic composite and the composite produced thereby useful for bone substitution. The composite can form its own substitutive,or in other words, it can act as a good osteoinductive as well as osteoconductive material when used in an orthopaedic surgery. Thus it has a potential use as an orthopaedic appliance in the medical field for the treatment of bone fracture.Bone substitute materials cover broadly all non-osseous materials implanted for such indications as acute fractures, delayed healirig fractures, pseudoarthroses etc. in order to promote the regeneration of bone. These are basically temporary substitutes which undergo complete degradation when used in vivo and promote ingrowth of bones. As reported by Chappard (Biomaterials, vol.14, no. 7. 507, 1993), the aim of bone grafting is basically to restore functional activiry of bones, enabling them to perform their biomechanical role and hence a bone graft or implant should ideally be biocompatible, osteoconductive and osteoinductive. Autografts and allografts have generally been in use for filling up bone defects. Katthagen (Bone regeneration with bone substitutes, Bemd-Dictrich-Kathagen-Springer publication. Chapter 3, p-18) has reported that autogenic cortical and spongy bones have initially been used as substitutes for bones. A.s reported by Burchardt (Journal of bone and Joint Surgery, 57, 814,1975), the autogenic cortical bones were less encouraged due to their low content of osteogenic stem cells and poor vascularisation. Although the success story of autogenic spongiosa as a bone transplant material has made it very popular in clinical practices, the following major problems associated with it may not perhaps be overlooked. i) Risk associated with autografts include longer operation and anaesthesia, high blood loss, infection, damage to nerves and blood v essels and most importantly the post operative pain. ii) Reconstructive surgery sometimes requires large amount of transplant materials which may not be sufficiently available in childhood. ill) Bone growth plate is prone to be damaged during bone explantation, especially in children. The above limitations of autografts prompted the researchers to opt for allogenic transplantation. Chalmers (Journal of Bone & .Joint Surgey 160, 41.1959) used freeze dried allogenic bone transplants in intramuscular pouches of rats to prove its osseoinductive capacities. But the allografts run a risk of secondary infection of Hepatitis and HIV. Poor availability of the suitable allograft is another major constraint of this transplantation. Thus the concept of xenogenic transplantation became popular among the researchers as a potential alternative. Elves et al (Journal of Bone & Joint Surgery. 56, 331,1974) used xenogenic bone in animals and reported development of cytotoxic antibodies at the affected site. In the begening of 60's, Maatz et al. (Journal of Bone & Joint Surgery (AM) 36, 721, 1954) developed Keil Bone Splinters by processing splintered pig-bones, which were cleaned, deproteinated using 20% hydrogen peroxide at 370C for 48-120 hours, degreased and finally sterilized. Both cortical splinters and spongiosa splinters were developed by this method. Salama et al (Journal of Bone &. Joint Surgery 60, 262, 1978) reported that the cortical splinters exhibited poor vascularization and hence the spongiosa splinters were better accepted But the major drawback associated with these splinters was improper bridging of gaps at the fracture site because of irregular shape and less available surface area of the splinters. Moreover, antigenacity of these splinters was also reported by Salama et al (Journal of Bone & Joint Surgery 60, 262,1978) while using the same in human being due to incomplete deproteination of the splinters. The limitation of incomplete deproteination of bones was overcome in the late seventies by completely deproteinating the same, as reported by Zitter ( Bone regeneration with bone substitutes, Chapter 3, p-26, Bemd-Dictrich-Kathagen-Springer pulication) , who charred the organic components of the bones resulting in disaggregation of crystalar structure and the product was known as Bone pyrost. The limitaton of this pyrost is that the organic components are completely burnt leading to shrinkage in bone trabeculae thereby widening the gap between the meshes and thus the bone formation takes place only on the surface, thereby hindering the complete penetration of the implant. Bohne ( Celle and materials, vol.3, no.4, p-377-382,1993) has reported that the calcium phosphate composites exhibit good osseoinductive property, but the resorption rate varies widely among its different available forms. lirist et al (Clinical orthopaedics, 187,111, 1984) showed that the combination of tncalcium phosphate salt with bone matrix resulied in twelve fold increase in bone formation compared to bone matrix alone. This was then followed by an era of usage of pure ceramics. Getter et al (Journal of Oral Surgery, 30, 263, 1972) studied 3 different calcium phosphate powders-tricalcium ceramics,calcium phosphate and hydraled calcium phosphate. The porosity was varied to provide improvement in osteo mduction Jarcho et al. (Clinical Orthopaedics, 157, 259, 1981) proved excellent compatability of the ceramic materials by varying their porosity. But the ceramic substitutes have the following limitations 1. Resorption rates are prolonged resulting in delayed healing. 2. Availability is low. 3. Very costly. Attention of the researchers gradually shifted towards skin-collagen based composite for using as bone substitute. Mittlemier (Bone regeneration with bone substitutes, Chapter 4, p-42, Bemd-Dictrich-Kathagen-Springer pulication,1977) developed a new biodegradable material called Collopat from penupharm pig skin. Collagen web was prepared by digesting the skin with trypsin and then freeze drying. Formaldehyde was used to reconstitute the collagen web and this was stirred with calcium phosphate before lyophilising the final product Collagen was crosslinked with formaldehyde in order to improve the incorporation of calciimi phosphate into the collagen mesh. Collopat developed from pentapharm pig skin showed unequivocal osteoregeneration due to poor agglomeration at the comers. Good regenerative effect was not observed in the composite. Moreover, the use of formaldehyde is not considered to be a desirable option from the hygiene point of view. Buckwalter (Climcal Orthopaedics, 112, 207-232, 1983) studied proteoglycan structure in calcifying cartilage and observed that the glycosaminoglycans inhibit crystal growth of calcium phosphate on collagen matrix. It has been reported by Roach (Cell Biology International, vol.l8,no.6, 617-628, 1994) that while some of the non-coUagenous proteins like osteopontin, osteonectin inhibit the crystal growth of calcium phosphate on the collagen matrix, sialoproteins and osteocalcin are known to accelerate the same. Researchers have shown that removal of the non-collagenous proteins from bone may remove some of the growth factors, thereby reducing the osseoinductive capacity of the material. It has been observed by various researchers that bone is an unstable tissue which undergoes constant remodelling by resorption and regeneration. As reported by Zitter (Bone regeneration with bone substitutes, Chapter 3, p-26, Bemd-Dictrich-Kathagen-Springer pulication), proper healing of bone fracture requires incorporation of a bone transplant, which in its initial phase gets degraded and is replaced by regenarated bone by a process called creeping substitution. This implies that the bone implant is expected to have high porosity and exhibit vascularisation. It is also supposed to offer scaffold for differentiation of bone cells. Further, the implant may have calcium phosphate with organic matrix to improve bone growth. No prior art is available on a bone implant exhibiting all these properties. The main objective of the present invetion is to provide a process for the preparation of novel bio-inorganic composite useful for bone substitution, which obviates the drawbacks stated above. Another objective of the present invention is to provide a new composite that resembles the substitutive tissue in form, structure, physical properties and function. Still another objective of the present invention is to facilitate growth of phase transformed calcium phosphate crystal on a preformed collagen matrix to ensure better adsorption and activation of the bone cells. Yet another obective of the present invetion is to provide a process for extracting protein building blocks from the implant surface for the synthensis of organic bone matrix. Still another objective of present invetion is to provide a process for making a composite which will offer scaffold for differentiation of bone cells. Accordingly the present invetion provides a process for the preparation of novel bio-inorganic composite useful for bone substitution which comprises: (i) washing cancellous rich bones by conventional manner using buffer saline and water successively, (ii) degreasing the bones by defattening solvents such as herein described in multiples stages for 8-11 hrs , wherein amount of solvent is 100% w/v of the weight of washed bone, (iii) conditioning the defatted bones with 4-8 times w/v aqueous organic solvent medium, such as herein described , over a period of 6-8 hrs., (iv) removing proteoglycans by treating the conditioned bones, as formed in step(iii), with a dissociative extracting solution selected from urea, guanidium monochloride, calcium chloride in the range of 20-50 % aqueous solution of 200- 400% w/w of the proteoglycan , over a period of 12-24 hrs. at pH in the range of 5.6-5.8 , at temperature ranging 4-20° C, (v) demineralization of the bones using demineralizing agents such as herein described, at pH in the range of 1-5 over 48-96 hrs., (vi) removing non/collagenous proteins as an optional step, using 7-12 times w/v of a conventional dissociative extracting solution as defined above over a period of 48-72 hrs at pH 7.2-7.4, (vii) lyophilizing the collagen matrix by the conventional manner at -70°C, (viii) treating the denatured collagen matrix, as prepared in step (vii), with saturated calcifying solution 1 - 3 % w/v dissolved in acidic water at a pH ranging 1 - 2.5 , followed by adjusting at pH in the range of 4.5 - 8.5 to get phase transformed calcium phosphate crystals and incorporating to it an antibiotic as defined herein at a range 0.25 - 1.25 % v/v , and keeping for 4-8 days at 30-37°C, ( ix) lyophilizing the resulting composite, as formed in step (viii) followed by sterilization by conventional way to get desired bio - inorganic composite. In an embodiment of the present invention, the source of the bones may be such as dead cow, buffalo, pig, collected from slaughter house ' . In another embodiment of the present invention, the pH used for washing the bones may be in the range of 6.5-7.5. In yet another embodiment of the present invention, defattening solvents used for degreasing the bones and conditioning the defatted bones may be methanol, acetone, diethyl ether, chlorinated solvents such as chloroform, dichloromethane, in different combinations. In still another embodiment of the present invention, the amount of organic solvents used for degreasing the bones may be minimum 100% w/v, on the weight of washed bones. In yet another embodiment of the present invention, the dissociative extracting solution used for removing the proteoglycans may be prepared from such as urea, guanidium monochloride,calcium chloride. In still another embodiment of the present invention, the amoimt of the dissociative extracting solution used for removing the proteoglycans may be in the range of 20-50% aqueous solution of 200-400% w/w, of the proteoglycan removing agent on the weight of the conditioned bones. In yet another embodiment of the present invention, the acid used for the demineralisation of the bones may be such as hydrochloric acid, citric acid, ethylene diamine tetra acetic acid (EDTA), ascorbic acid. In still another embodiment of the present invention, the phase transformed calcium phosphate crystals may be such as brushite, octacalcium phosphate, hydroxyapatite or their intermediaries. In yet another embodiment of the present invention, the amount of saturated calciying solution added to the preformed collagen matrix may be 15-25 times w/v, on the weight of the collagen matrix. In still another embodiment of the present invention, antibiotics used may be such as kanamycin, streptomycin, tetracycline, amoxyciline, gentamycin, clotrimazole, mycanazole, either individually or in combination. In yet another embodiment of the present invention, the amount of antibiotics used may be in the range of 0.5-1.25% v/v, on the saturated calcifying solution. Accordingly the present invention provides a novel bio-inorganic composite prepared by the process as described above. Bones rich in cancellous component are washed in a known method at a temperature in the range of 4-20°C for about 24-72 hrs to remove the cell debris, adhering blood components and other extraneous tissue components. These are then degreased in multi stage by sttirring with minimum 100% w/v of organic solvent on the weight of bone ata temperature in the range of 4-200C over a period of 8-11 hrs. till the fat content of the bone comes down below 5%. These defatted bones are conditioned over a period of 6-8 hrs. by stirring with 4-8 times MVV, of an aqueous solution of an organic solvent followed by 4-8 times w/v, of plain water wash. The proteoglycans present in the bones are then removed at 4-20°C by dissociative extraction method lising 20-50% aqueous solution of 200-400% w/w, of a proteoglycan removing agent on the weight of the conditioned bones, over a period of 12-24 hours at a pH in the range of 5.6-5.8. The bones are then demineralised by acid treatment at a pH in the range of 1-5 over a period of 48-96 hours. Non-collageneous proteins present in the substrate are removed as an optional step by known method using 7-12 times w/v, of a conventional dissociative extracting solution over a period of 48-72 hrs at pH in the range of 7.2- 7.4. Then the sample is lyophilised in a conventional way to get denatured collagen matrix ready for crystal growth. l-3%,w/v, Dicalcium phosphate is dissolved in acidified water at a pH in the range of 1-2.5 to get a saturated solution and the pH is subsequently adjusted in the range of 4.5-8.5 to get the different phase transformed calcium phosphate crystals. The solution is filtered to get clear calcifying solution and 0.25-1.25% v/v, of an antibiotic, dissolved in 2-5 times of water, is added to it with vigorous stirring. The denatured collagen matrix is added to the calcifying solution incorporated with antibiotic and the system is kept at 30-37°C for 4-8 days. The resulting bio-inorganic composites are lyophilised in conventional way and sterilised by a known method after cutting into desired size and shape. The novelty of the present tnveotion lies in the selection of cancellous rich bones, providing a method for preparing denatured collagen matrix suitable for crystal growth and facilitating in vitro crystal growth on the matrix by using phase transformed calcium phosphate crystals resulting in a product resembling natural bone in form, structure and function. 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 10 pieces of femur bones of cow were collected from slaughter house. They were slit longitudinally with an electric burr and inner soft bone or cancellous bone was removed using scalpel and forceps. These cancellous bones were cut into pieces of about 2x6 cm rectangular blocks. 104 gms of these spongiosa pieces were taken in a beaker containing phosphate buffered saline maintained at pH 6.5 and stirred with a magnetic stirrer at 20°C for about 48 hrs. to remove the cell debris, adhering blood components and other extraneous components. Then the pieces were washed in two changes of water and taken in a 1lit. beaker for degreasing. 300 ml chloroform was added to the bones and after stirring for 3 hrs. the liquid in the beaker was replaced by a mixture of 200 ml chloroform and 100 ml methanol and stirring was continued After 2 hrs, the liquid in the beaker was drained out and a mixture of 150ml chloroform and 150 ml methanol was added to the beaker with stirring. After another 2 hours the liquid was again drained and the bones were finally degreased by stirring with 300 ml pure methanol over 2.5 hours. Soxhlet extraction confirmed the absence of any fat in the bones. Weight of these degreased bones was found to be 70 gms., which were stirred with a mixture of 150 ml methanol and 150 ml water for 4 hrs. Finally the bones were washed with 300 ml distilled water at 20°C for 3 hrs. Weght of the bones at this stage was found to be 35gms. 72 gms urea was added to 300 ml water and the degreased bones were stirred at 4°C in this solution, pH of which was adjusted at 5.8 by adding 4-5 drops of 0.05 M TRIS, to remove the proteoglycans present in the bones. After 12 hrs. the bone pieces were taken out in a separate beaker and 300ml of 0.6 N HCl was added to it. pH was noted as 1. After 72 hrs. the bones were taken out in another beaker. 300 ml of an aqueous mixture of 114.63 gms of Guanidium monochloride and 43.83gms EDTA was added to it with stirring and pH was adjusted to 7.2 by adding 3 drops of 0.05M tris- hydroxymethyl methylamine (TRIS). Stirring was continued for 48 hrs with two mtermediate washings to remove the non-collagenous proteins present in the bones. The denatured collagen matrix, {Miepared thus, was lyophilised at -70°C and 120 M torr. 2 gms dicalcium phophate salt (CaHPO4,2 H2O) was dissolved in 100 ml water and the pH was adjusted at 2 using 0.5M HCl. The pH was subsequently raised to 4.5 using 0.5M KOH to prepare calcifying solution of brushite. An aqueous antibiotic solution was then prepared by adding 0.5 ml amoxycillin to 2 ml water and this was added to the calcifying solution to prevent bacterial growth. 50 ml of this calcifying solution was added to the beaker containing 2.49 gms of the preformed collagen matrix and 4.6 mg of seed crystal of brushite was also added to it. The whole system was kept in a dessicator for 4 days at 30°C. The bio-inoganic composites, so formed after the crystallisation of brushite on the collagen matrix, were lyophilised at -70°C and 120 M torr. Finaly, the composites were cut into desired size and shape and were sterilized by gamma irradiation at 2 M rads and packed in polythene covers. Example II 10 pieces of femur bones of buffalo were collected from slaughter house. They were slit longitudinally with an electric burr and inner soft bone or cancellous bone was removed using scalpel and forceps. These cancellous bones were cut into pieces of about 2 X 6 cm rectangular blocks. 150 gms of these spongiosa pieces were taken in a beaker containing phosphate buffered saline maintained at pH 7 and stirred with a magnetic stirrer at 4°C for about 72 hrs. to remove the cell debris, adhering blood components and other extraneous components. Then the pieces were washed in two changes of water and taken in a 1lit. beaker for degreasing. 350 ml chloroform was added to the bones and after stirring for 4 hrs. the liquid in the beaker was replaced by a mixture of 200 ml chloroform and 100 ml methanol and stirring was continued. After 3.5 hrs, the liquid in the beaker was drained out and the bones were finally degreased by stirring with 300 ml pure methanol over 3.5 hours. Soxhlet extraction confirmed the absence of any fat in the bones. Weight of these degreased bones was found to be 90 gms., which were stirred with a mixture of 200 ml methanol and 200 ml water for 5 hrs. Finally the bones were washed with 400 ml distilled water at 4°C for 2 hrs. Weght of the bones at this stage was found to be 50gms. 66.87 gms guanidine monochloride was added to 350 ml water and the degreased bones were stirred at 10°C in this solution, pH of which was adjusted at 5.6 by adding 6 drops of 0.05 M TRIS, to remove the proteoglycans present in the bones. After 18 hrs. the bone pieces were taken out m a separate beaker and 300ml of 0.5 M EDTA was added to it, pH was noted as 3. After 96 hrs. the bones were taken out in another beaker. Non-collagenous proteins wee allowed to remain as such in the bone The denatured collagen matrix, prepared thus, was lyophilised at -70°C and 120 M torr, 1 gm dicalcium phophate salt (CaHPO4,2 H2O) was dissolved in 100 ml water and the pH was adjusted at 2 using 0.5M HCl. The pH was subsequently raised to 7.3 using 0.5M KOH to prepare calcifying solution of octacalcium phosphate. An aqueous antibiotic solution was then prepared by adding 0.5 ml streptomycin to 2 ml water and this was added to the calcifying solution to prevent bacterial growth. 50 ml of this calcifying solution was added to the beaker containing 2.49 gms of the preformed collagen matrix and 4mg of seed crystal of octacalcium phosphate was also added to it. The whole system was kept in a dessicator for 7 days at 32°C. The bio-inoganic composites, so formed after the crystalHsation of octacalcium phosphate on the collagen matrix, were lyophilised at -70°C and 120 M torr. Finaly, the composites were cut into desired size and shape and were sterilized by gamma irradiation at 2 M rads and packed in polythene covers. Example III 10 pieces of femur bones of pig were collected from slaughter house. They were slit longitudinally with an electric burr and inner soft bone or cancellous bone was removed using scalpel and forceps. These cancellous bones were cut into pieces of about 2x6 cm rectangular blocks. 175 gms of these spongiosa pieces were taken in a beaker containing phosphate buffered saline maintained at pH 7.5 and stirred with a magnetic stirrer at 10°C for about 24 hrs. to remove the cell debris, adhering blood components and other extraneous components. Then the pieces were washed in three changes of water and taken in a Hit. beaker for degreasing. 350 ml chloroform was added to the bones and after stirring for 4 hrs. the liquid in the beaker was replaced by a mixture of 200 ml chloroform and 200 ml methanol and stirring was continued. After 3 hrs, the liquid in the beaker was drained out and the bones were finally degreased by stirring with 400 ml pure methanol over 3.5 hours. Soxhlet extraction confirmed that the fat content of the bones was 5%. Weight of these degreased bones was found to be 120 gms., which were stirred with a mixture of 100 ml methanol and 300 ml water for 4 hrs. Finally the bones were washed with 400 ml distilled water at 10°C for 2 hrs. Weght of the bones at this stage was found to be 80gms. 66.87 gms guanidine monochloride was added to 350 ml water and the degreased bones were stirred at 20°C in this solution, pH of which was adjusted at 5.6 by adding 6 drops of 0.05 M TRIS, to remove the proteoglycans present in the bones. After 12 hrs. the bone pieces were taken out in a separate beaker and 300ml of 0.5 M citric acid was added to it. pH was noted as 4 After 72 hrs. the bones were taken out in another beaker. 300 ml of an aqueous mixture of 114.63 gms of Guanidium monochloride and 43.83gms EDTA was added to it with stirring and pH was adjusted to 7.4 by adding 3 drops of 0.05M TRIS. Stirring was continued for 72 hrs with two intermediate washings to remove the non- collagenous proteins present in the bones. The denatured collagen matrix, prepared thus, was lyophilised at -70°C and 120 M torr. 3gms dicalcium phophate salt (CaHPO4,2 H2O) was dissolved in 100 ml water and the pH was adjusted at 2 using 0.5M HCl. The pH was subsequently raised to 8.2 using 0.5M KOH to prepare calcifying solution of hydroxyapatite. An aqueous antibiotic solution was then prepared by adding 0.5 ml tetracycline to 2 ml water and this was added to the calcifying solution to prevent bacterial growth. 50 ml of this calcifying solution was added to the beaker containing 4 gms of the preformed collagen matrix and 4mg of seed crystal of hydroxyapatite was also added to it. The whole system was kept in a dessicator for 5 days at 35°C. The bio-inoganic composites, so formed after the crystallisation of hydroxyapatite on the collagen matrix, were lyophilised at -70°C and 120 M torr. Finaly, the composites were cut into desired size and shape and were sterilized by gamma irradiation at 2 M rads and packed in polythene covers. Example IV 10 pieces of femur bones of cow were collected from slaughter house. They were slit longitudinally with an electric burr and inner soft bone or cancellous bone was removed using scalpel and forceps. These cancellous bones were cut into pieces of about 2x6 cm rectangular blocks. 120 gms of these spongiosa pieces were taken in a beaker containing phosphate buffered saline maintained at pH 7.3 and stirred with a magnetic stirrer at 20°C for about 48 hrs. to remove the cell debris, adhering blood components and other extraneous components. Then the pieces were washed in two changes of water and taken in a Hit. beaker for degreasing. 300 ml chloroform was added to the bones and after stirring for 2.5 hrs. the liquid in the beaker was replaced by a mixture of 200 ml chloroform and 100 ml methanol and stirring was continued. After 2 hrs, the liquid in the beaker was drained out and a mixture of 150ml chloroform and 150 ml methanol was added to the beaker with stirring. After another 2 hours the liquid was again drained and the bones were finally degreased by stirring with 300 ml pure methanol over 2hours. Soxhlet extraction confirmed that the fat content of the bones was 1%. Weight of these degreased bones was found to be 70 gms., which were stirred with a mixture of 150 ml methanol and 150 ml water for 4 hrs. Finally the bones were washed with 300 ml distilled water at 20°C for 3 hrs. Weght of the bones at this stage was found to be 35gms. 72 gms urea was added to 300 ml water and the degreased bones were stirred at 15°C in this solution, pH of which was adjusted at 5.8 by adding 4-5 drops of 0.05 M TRIS, to remove the proteoglycans present in the bones. After 16 hrs. the bone pieces were taken out in a separate beaker and 300ml of 0.6 N HC1 was added to it. pH was noted as 1. . Stirring was continued for 48 hrs. Non-collagenous proteins wee allowed to remain as such in the bone.The denatured collagen matrix, prepared thus, was lyophilised at -70°C and 120 M torr. 2 gms dicalcium phophate salt {CaHPO4,2 H2O) was dissolved in 100 ml water and the pH was adjusted at 2 using 0.5M HC1. The pH was subsequently raised to 6.5 using 0.5M KOH to prepare calcifying solution of intermediary phase of conversion of brushite to octacalciun phosphate. An aqueous antibiotic solution was then prepared by adding a mixture of 0.25 ml clotrimazoleand 0.25 ml.mycanazole to 2 ml water and this was added to the calcifying solution to prevent bacterial growth. 50 ml of this calcifying solution was added to the beaker containing 3 gms of the preformed collagen matrix. The whole system was kept in a dessicator for 6 days at 30°C.. The bio-inoganic composites, so formed after the crystallisation of the intermediary phase on the collagen matrix, were lyophilised at -70°C and 120 M torr. Finaly, the composites were cut into desired size and shape and were sterilized by gamma irradiation at 2 M rads and packed in polythene covers. Example V 10 pieces of femur bones of buffalo were collected from slaughter house. They were slit longitudinally with an electric burr and toner soft bone or cancellous bone was removed using scalpel and forceps. These cancellous bones were cut into pieces of about 2 X 6cm rectangular blocks. 150 gms of these spongiosa pieces were taken in a beaker containing phosphate buffered saline maintained at pH 6.9 and stirred with a magnetic stirrer at 4°C for about 72 hrs. to remove the cell debris, adhering blood components and other extraneous components. Then the pieces were washed in two changes of water and taken in a 11it. beaker for degreasing. 350 ml chloroform was added to the bones and after stirring for 4 hrs. the liquid in the beaker was replaced by a mixture of 200 ml chloroform and 100 ml methanol and stirring was continued. After 3 hrs, the liquid in the beaker was drained out and the bones were finally degreased by stirring with 300 ml pure methanol over 3.5 hours. Soxhlet extraction confirmed the absence of any fat in the bones. Weight of these degreased bones was found to be 90 gms., which were stirred with a mixture of 200 ml methanol and 200 ml water for 5 hrs. Finally the bones were washed with 400 ml distilled water at 4°C for 3 hrs. Weght of the bones at this stage was found to be 50gms. 67.8 gms calcium chloride was added to 300 ml water and the degreased bones were stirred at 20°C in this solution, pH of which was adjusted at 5.6 by adding 6 drops of 0.05M TRIS, to remove the proteoglycans present in the bones. After 24 hrs. the bone pieces were taken out in a separate beaker and 300ml of 0.5 M EDTA was added to it. pH was noted as 3. After 96 hrs. the bones were taken out in another beaker. Non-collagenous proteins were allowed to remain as such in the bone. The denatured collagen matrix, prepared thus, was lyophilised at -70°C and 120 M torr. 1 gm dicalcium phophate salt (CaHPO4,2 H2O) was dissolved in 100 ml water and the pH was adjusted at 2 using 0.5M HCl The pH was subsequently raised to 7.6 using 0.5M KOH to prepare calciiying solution of intermedial}' phase of conversion of octacalciim phosphate to hydroxyapatite. An aqueous antibiotic solution was then prepared by adding 0.5 ml gentamycin to 2 ml water and this was added to the calcifying solution to prevent bacterial growth. 50 ml of this calcifying solution was added to the beaker containing 3.5 gms of the preformed collagen matrix. The whole system was kept in a dessicator for 8 days at 37°C. The bio-inoganic composites, so formed after the crystallisation of intermediary phase on the collagen matrix, were lyophilised at -70°C and 120 M torr. Finaly, the composites were cut into desired size and shape and were sterilized by gamma irradiation at 2 M rads and packed in polythene covers. The main advantages of the present inveatlon are the following:-1) The present invention provides a method of utilising bones, which are important byproducts of slaughterhouse, for very useful purpose of bone transplantation. 2) The new implant resembles the substitutive natural bone tissue in form, structure, physical properties and fimctional requirements 3) This hew composite is spongy in nature and acts as an excellent scaffold for differentiation of bone cells towards vascularization and facilitates influx of cells at the fracture site as the first stage of healing 4) Its porosity permits faster fonnation of granulation tissue and regeneration of connective tissue and bone. 5) Hydroxyapatite or calcium phosphate in the material acts as an amphoteric ion exchanger. Selective appearance of calcium and phosphate ions occur and as a consequence negative charge is induced on the its surface. This leads to the formation stimulation for the formation of new bone. 6) The implant can be easily shaped accordmg to the requirement of the defective site. 7) Antibiotics or growth factor can be entrapped on to the matrix and can be used at the defective site. We claim 1. A process for the preparation of novel bio-inorganic composite useful for bone substitution which comprises: i) washing cancellous rich bones by conventional manner using saline buffer and water successively, ii) degreasing the bones by defattening solvents such as herein described in multiples stages for 8-11 hrs , wherein amount of solvent is 100% w/v of the weight of washed bone, iii) conditioning the defatted bones with 4-8 times w/v aqueous organic solvent medium , such as herein described , over a period of 6-8 hrs., iv) removing proteoglycans by treating the conditioned bones, as formed in step(iii), with a dissociative extracting solution selected from urea, guanidium monochloride, calcium chloride in the range of 20-50 % aqueous solution of 200-400% w/w of the proteoglycan , over a period of 12-24 hrs. at pH in the range of 5.6-5.8 , at temperature ranging 4-200C, v) demineralization of the bones using demineralizing agents such as herein described, at pH in the range of 1-5 over 48-96 hrs., vi) removing non/collagenous proteins as an optional step, using 7-12 times w/v of a conventional dissociative extracting solution as defined above over a period of 48-72 hrs at pH 7.2-7.4, vii) lyophilizing the collagen matrix by the conventional maimer at -700C, viii) treating the denatured collagen matrix, as prepared in step (vii), with saturated calcifying solution 1 - 3 % w/v dissolved in acidic water at a pH ranging 1 - 2.5 , followed by adjusting at pH in the range of 4.5 - 8.5 to get phase transformed calcium phosphate crystals and incorporating to it an antibiotic as defined herein at a range 0.25 - 1.25 % v/v , and keeping for 4-8 days at 30-370C, ix) lyophilizing the resulting composite, as formed in step (viii) followed by sterilization by conventional way to get desired bio - inorganic composite. 2. A process, as claimed in claim 1, wherein the source of the bones is dead buffalo, pig, collected from slaughter house .. 3. A process, as claimed in claims 1 & 2, wherein the pH of saline buffer used for washing the bones is in the range of 6.5-7.5. 4. A process, as claimed in claims 1 to 3, wherein the organic solvents used for degreasing the bones and conditioning the defatted bones are such as methanol, acetone, di-ethyl ether, chlorinated solvents such as chloroform, dichloromethane, in different combinations. 5. A process, as claimed in claims 1 to 6, wherein the demineralising agents used are such as hydrochloric acid, citric acid, ethylene diamine tetra acetic acid (EDTA) citric acid, ascorbic acid. 6.A process, as claimed in claims 1 to 7, wherein the phase transformed calcium phosphate crystals are such as brushite, octacalcium phosphate, hydroxyapatite or their intermediaries. 7.A process, as claimed in claims 1 to 9, wherein antibiotics used is selected from kanamycin, streptomycin, tetracycline, amoxyciline, gentamycin, clotrimazole, mycanazole, either individually or in combination. i. A process for the preparation of novel bio-inorganic composite useful for bone substitution substantially as herein described with reference to the examples.

Documents:

3065-del-1998-abstract.pdf

3065-del-1998-claims cancelled.pdf

3065-del-1998-claims.pdf

3065-del-1998-complete specification(granted).pdf

3065-del-1998-correspondence-others.pdf

3065-del-1998-correspondence-po.pdf

3065-del-1998-description (complete).pdf

3065-del-1998-form-1.pdf

3065-del-1998-form-2.pdf

3065-del-1998-form-3.pdf

3065-del-1998-form-4.pdf

3065-del-1998-form-9.pdf