Patent Application: US-22433894-A

Abstract:
a method of preparing a delivery system for a biologically active growth and morphogenetic factor by using chromatographic techniques and which includes introducing such biologically active factor into a chromatography column of appropriate dimensions which contains an integral porous carrier comprising a solid adsorbent selected for its specific affinity for such biologically active factor . in one embodiment of the invention , porous hydroxyapatite is used as the carrier for bone morphogenetic proteins . the invention extends to a delivery system prepared according to the above method ; to an apparatus for preparing the delivery system according to this method ; and to use of the delivery system in a method for inducing the information of new bone in primates .

Description:
it should be understood that it is solely for descriptive purposes that the invention is illustrated using bmp fractions isolated from the extracellular matrix of baboon bone and adsorbed on porous hydroxyapatite carrier discs , 25 mm in diameter and 3 . 5 mm in thickness , and that the invention should not be construed as being limited thereto . referring now to fig1 - 7 of the drawings , fig1 shows biologically active bmps fractions , as determined by alkaline phosphatase activity and histology , being loaded onto porous hydroxyapatite carrier discs 14 placed within a standard column for liquid chromatography . loading and subsequent adsorption of bmps fractions is facilitated by a modification of the column inlet 18 . this modification consists of connecting the column inlet 18 to a capillary tube 20 , 2 μs in diameter , in plastic or glass to the top end sealing piece 22 of the column . the level 24 of the starting buffer ( 5 mm hcl ) is regulated so as to be 10 mm above the tip 26 of the capillary tube 20 . fig2 shows a photomicrograph of a central area of the osteogenic delivery system . there is extensive bone formation within the porous spaces of the carrier disc harvested 30 days after intramuscular implantation in a male baboon . white empty spaces represent the hydroxyapatite framework dissolved after histological processing and decalcification of the specimen . fig3 shows a photomicrograph of a peripheral area of the osteogenic delivery system . there is bone formation within the porous spaces of the carrier disc harvested 30 days after intramuscular implantation in a male baboon . note the muscular tissue ( left ) surrounding the osteogenic delivery system . fig4 shows a high power photomicrograph of the osteogenic delivery system . there is extensive bone formation within the porous spaces of the carrier disc . note the pronounced cellularity and vascularity of the newly formed bone . fig5 shows a photomicrograph of a central region of a porous hydroxyapatite disc implanted in a male baboon without prior adsorption of bmps fractions . note the limited cellular and vascular invasion within the porous spaces and the lack of bone formation . fig6 shows a chromatographic profile of bmps fractions on porous hydroxyapatite discs . arrow indicates loading of the biologically active bmps fractions in 5 mm hcl . fig7 shows a schematic representation of the bioassays performed to test the osteogenic property of the delivery system . arrow a indicates the point at which bmps fractions are taken , before chromatography , for testing in the subcutaneous space of the rat . arrow b indicates that the osteogenic delivery system is taken after adsorption chromatography of bmps fractions for intramuscular placement in primates . arrow c indicates that unbound material ( eluate ) is taken after the chromatography step for placement in the subcutaneous space of the rat . integral implant carriers consisting of porous nondegradable hydroxyapatite were obtained in disc form , 25 mm in diameter and 3 . 5 mm in thickness . the solid components of the framework average 130 μm in diameter and their interconnections average 220 μm in diameter . the average porosity is 600 μm and their interconnections average 260 μm in diameter ( interpore 500 , interpore international , irvine , calif ., usa ). the hydroxyapatite is obtained after hydrothermal chemical exchange with phosphate converting the original calcium carbonate exoskeletal microstructure of the scleractinian reef - building coral of the genus goniopora ( 5 ) into an inorganic replica of hydroxyapatite ( 6 - 8 ). conversion to hydroxyapatite is monitored by x - ray diffraction pattern , showing that hydroxyapatite replicas consist of 90 percent hydroxyapatite and 10 percent tricalcium phosphate . dehydrated diaphyseal baboon bone matrix , sieved to a discrete particle size of 74 - 420 μm , was demineralized in 0 . 5n hcl and dissociatively extracted in 4m guanidine - hcl , 50 mm tris , ph 7 . 4 , containing protease inhibitors ( 1 ). baboon bmps were isolated using methods in reference 1 , and as described for the purification of bmp - 3 ( osteogenin ) from baboon bone matrix ( 4 ). after gel filtration chromatography , bmp fractions were tested as follows , for biological activity in the subcutaneous space of a rat . osteogenic activity is tested by reconstituting 25 mg of rat insoluble collagenous bone matrix ( the inactive carrier obtained after dissociative extraction of rat bone matrix ) with 5 to 20 μg of bmp fractions as described ( 1 , 4 ). after implantation in the subcutaneous space of the rat , implants were harvested at day 11 and examined biochemically ( alkaline phosphatase activity ) and histologically . controlled adsorption and binding of bmp fractions onto hydroxyapatite carrier : creation of the osteogenic delivery system 1 ) adsorption of bmps onto porous hydroxyapatite carrier using liquid chromatography techniques to provide a delivery system according to the invention , and 2 ) the use of this delivery system to delivery bmps and to induce the formation of new bone after in vivo implantation in primates . the most active bmp fractions , as determined by alkaline phosphatase activity and histological evidence of florid bone formation in the subcutaneous space of the rat , were concentrated and exchanged with 5 mm hydrochloric acid ( hcl ) using ultrafiltration membranes with 10 , 000 mw cutoff ( amicon , ym - 10 ). prior to adsorption onto hydroxyapatite discs , aliquots of osteogenin fractions in 5 mm hcl were tested for biological activity in the subcutaneous space of the rat . in an apparatus 10 according to the invention , a liquid chromatography column 12 with an internal diameter of 25 mm , was loaded with porous hydroxyapatite carrier discs 14 , 25 mm in diameter ( fig1 ). the column was equilibrated with starting buffer ( 5 mm hcl ). after equilibration , bmp fractions , dissolved in 5 ml of starting buffer 16 , were loaded onto the porous hydroxyapatite carrier discs 14 , thereby providing delivery systems according to the invention for implantation . loading and subsequent adsorption of bmp fractions was facilitated by a modification of the column inlet 18 . this modification comprised connecting the column inlet 18 to a capillary tube 20 , 2 mm in diameter , in plastic or glass located through the top end sealing piece 22 of the column 12 . the level 24 of the starting buffer was regulated so as to be 10 mm above the tip 26 of the capillary tube 20 . after loading , the capillary outlet 28 of the column 12 was connected with the column inlet providing a continuous recirculation of starting buffer and unbound material ( fig1 ). reference numeral 30 indicates the eluate . in a preferred embodiment , at least a portion of the eluate 30 from outlet 28 is diverted for analysis 40 in order to determine the unbound fraction . the data may be recorded on an appropriate recording device 42 . after stabilization of unbound fractions on the chromatographic profile ( fig6 ), unbound fractions were collected in 20 ml of starting buffer ( column capacity ) for testing their biological activity in the subcutaneous space of the rat in the manner described above . after recovery of the unbound material in starting buffer , the bmps - hydroxyapatite carrier discs 14 were left to dry in a laminar flow cabinet at room temperature . 2 . in vivo bioassay of the osteogenic delivery system ( fig7 ) the in vivo bioassay consisted of the following three steps : a ) bioassay of the bmp fractions before loading the hydroxyapatite carrier discs ; b ) bioassay of the osteogenic delivery system in primates of the genus papio ( baboon , papio ursinus ); and c ) bioassay of unbound protein fractions to determine residual biological activity , if any , in the unbound material . a ) bmp fractions were tested for biological activity as ; described above under the heading &# 34 ; purification and preparation of baboon bmp fractions &# 34 ;. b ) bmps - hydroxyapatite carrier discs were implanted intramuscularly in male baboons to test the osteogenic activity of the delivery system . bone formation within the hydroxyapatite discs were assessed histologically after harvesting the implants 30 days after surgical implantation . for the purpose of testing its osteogenic potential , the delivery system was implanted in extraskeletal sites to provide unequivocal proof that any bone formation was induced by bmps delivered during healing and incorporation , by the hydroxyapatite carrier . fig2 shows extensive bone formation within the porous spaces of a hydroxyapatite carrier disc after adsorption chromatography of biologically active bmp fractions , and harvested 30 days after being implanted intramuscularly in a male baboon . the white empty spaces evident in fig2 represent the hydroxyapatite framework dissolved after histological processing and decalcification of the specimen . fig3 shows extensive vascular invasion and bone differentiation in a peripheral area of a hydroxyapatite carrier disc after adsorption chromatography of biologically active bmp factions , and harvested 30 days after being implanted intramuscularly in a male baboon . attention is drawn , in particular , to the muscular tissue ( left ) surrounding the osteogenic delivery system . fig4 shows details of the newly formed bone within the porous spaces of the hydroxyapatite carrier disc after adsorption chromatograph of biologically active bmp fractions . as can be seen , extensive bone formation has occurred within the porous spaces of the carrier disc . the pronounced cellularity and vascularity of the newly formed bone should be noted . discs of hydroxyapatite carriers were implanted without chromatographic adsorption of bmps , as control . fig5 shows limited penetration of fibrovascular and cellular elements and lack of bone differentiation . c ) possible residual biological activity in the unbound fraction collected from the column after adsorption chromatography was assessed by reconstituting 25 mg of rat insoluble collagenous carrier with increasing concentration of unbound protein fractions . no bone differentiation occurred in implants reconstituted with 50 , 200 , 500 , 1000 and 2000 μl respectively of unbound material . these results demonstrate the osteogenic properties of the invention after chromatographic adsorption of bmp fractions onto porous hydroxyapatite carriers and implantation in extraskeletal sites of primates . it will be understood that it is intended to cover all changes and modification of the description of the invention herein disclosed for the purpose of illustration which do not constitute departures from the spirit and scope of the invention . in particular , the chromatographic technique is not limited to the adsorption of bmps ( including both native and human recombinant material ), but extends to other biologically active cellular modulators such as laminin , type iv collagen , transforming growth factors beta , insulin growth factor ii , platelet derived growth factor , fibroblast growth factor ( s ), and growth hormone . in addition , the porous material used as carrier is not limited to hydroxyapatite replicas , but includes a variety of other porous biomaterials , such as sintered hydroxyapatite , polymers , plasma - 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