Patent Abstract:
an thermally sterilized bone paste useful in the orthopedic arts , for example in the repair of non - union fractures , periodontal ridge augmentation , craniofacial surgery , implant fixation , impaction grafting , or any other procedure in which generation of new bone is deemed necessary , is provided by a composition comprising a substantially bioabsorbable osteogenic compound in a matrix of 11 - 19 %, and preferably about 15 - 19 % or thermally sterilized gelatin . in various embodiments , the osteogenic compound is selected from demineralized bone matrix ; bioactive glass ceramic , bioglass ®, bioactive ceramic , calcium phosphate ceramic , hydroxyapatite , hydroxyapatite carbonate , corraline hydroxyapatite , calcined bone , tricalcium phosphate , or like material ; bone morphogenetic protein , tgf - β , pdgf , or mixtures thereof , natural or recombinant ; and mixtures of -.

Detailed Description:
it will be appreciated by those skilled in the art that the specifics of the composition of this invention , its method of preparation and use are applicable to such compositions for use in any vertebrate species . nonetheless , because human use is considered likely to be the principal orthopedic application of this new material , the following description concentrates on exemplifying this material for human applications . the composition of this invention comprises gelatin and additional osteogenic components . the gelatin is preferably thermally cross - linked at about 38 ° c ., at a gelatin concentration of between about 11 - 19 %, and preferably at between about 15 %- 19 % ( w / w ), and the osteogenic components are selected from : ( i ) demineralized bone , preferably derived from the species into which the thermally sterilized bone paste is to be implanted ; or ( ii ) bioactive glass ceramic , bioglass ®, bioactive ceramic , calcium phosphate ceramic , hydroxyapatite , hydroxyapatite carbonate , corraline hydroxyapatite , calcined bone , cortical bone chips , cancellous bone chips , tricalcium phosphate , like material , or mixtures thereof ; or ( iii ) bone morphogenetic protein , osteogenic proteins or peptides ( e . g . osteogenin , p15 , cdmp , and the like ), tgf - beta , pdgf , or mixtures thereof , natural or recombinant ; or the composition is fluid at a first temperature ( e . g ., above 38 ° c .) and becomes thermally cross - linked at or just above a second temperature , corresponding to the normal body temperature of the organism into which the composition is to be implanted ( e . g ., at 38 ° c . in humans ). the terms “ thermally cross - linked ” or “ thermally cross - linkable ” are used herein to describe the property of a composition which contains molecules which , at or below a given temperature and concentration , associate in such a fashion as to result in gellation of a solution containing these molecules . the term “ thermally sterilized ” is used herein to indicate that a material has been treated under such conditions of temperature as are generally recognized in the art to render a material sterile ( i . e . devoid of living organisms ). for example , the standard procedure of “ autoclaving ” a material occurs in a sealed chamber into which steam is pumped to such a pressure that the temperature within the chamber reaches approximately 121 ° c . treatment of twenty minutes under such conditions is generally recognized as being sufficient to surface sterilize an object , with longer periods being required , depending on the volume of an object or liquid , through which heat is to be transferred . other conditions of dry heat ( i . e . absent steam ) are also generally acknowledged as producing a sterile environment , as in , for example , approximately 121 - 130 ° c . of dry heat for from about a half hour to about six hours . once again , considerations of time of exposure are required in order to achieve a sterile filed . in any event , this term as applied to the composition of this invention does not require that the material described as “ thermally sterilized ” remain in a sterile state . in other words , the material may be implanted , in which case it would preferably remain sterile , or it may be left exposed on a shelf in an open and contaminated state , and yet still have been “ thermally sterilized ”. it is the physical characteristics of the thus - treated material ( i . e . molecular weight and solution behavior , as revealed by the kinematic viscosity ), that is critical , rather than the state of being sterile or not . according to this disclosure , treatment of a dried gelatin composition for approximately 0 . 5 - 18 hours , and preferably between about 3 - 6 hours at between about 121 ° c . to 130 ° c . is considered to come within the meaning of the term “ thermally sterilized ”. the term “ substantially bioabsorbable ” is used herein to describe the property of a material is a material which is no longer detectable at the site of implantation after a reasonable period of biological resorption , such as three months to a year later . accordingly , for example , demineralized bone matrix which has been chemically cross - linked with an agent such as glutaraldehyde , is not considered to be substantially bioabsorbable . however , demineralized bone matrix itself , gelatin , and bone morphogenetic factors are all considered to be substantially bioabsorbable as they cooperate in new bone formation , rather than purely providing structural rigidity or support . the gelatin acts as a carrier phase and has the ability to thermally cross - link over a very small temperature range . this thermal cross - linking reaction is largely controlled by physical entanglement and hydrogen bonding between chains , and so is dependant on concentration and temperature . ( sperling ). additionally , since gelatin has been used extensively in the medical market , its in vivo properties are thoroughly studied . ( mcdonald ). the gel - foam sponge is the most familiar application of this biopolymer . studies have indicated that gelatin is only mildly antigenic upon implantation , and is comparable in some of its properties to collagen , ( mcdonald ). however , collagen does not exhibit the thermal cross - linking property so important to the composition of this invention . where present , the bioactive glass , such as bioglass ®, bioactive ceramic , calcium phosphate ceramic , hydroxyapatite , hydroxyapatite carbonate , calcined bone , tricalcium phosphate , or like material , is included to enhance the range of manipulable characteristics of strength and osteogenesis ( osteoinduction and osteoconduction ) exhibited by the composition . the manufacture of gelatin is based on the partial hydrolysis of collagen . collagen is available from skin , bone , cartilage , tendon and other connective tissue . skin and bone yield type i and type iii collagen molecules , while tendon yields nearly pure type i collagen , and cartilage yields a mixture of type ii and rarer types of collagen molecules . gelatin molecules resemble collagen triple helices , however , they are partially hydrolyzed . as a result , in solution they have little organization . but , as the solution cools , the gelatin molecules begin to form helical structures . as the solution cools further , the viscosity increases and a phase transformation from a solution to a gel occurs . this phase change is reversible when heat is added . the set time and set temperature of a gelatin solution are dependent on the concentration of gelatin in solution , the molecular weight , or intrinsic viscosity , of the gelatin molecules , and the ph of the solution . at the isoelectric point , or the ph at which the gelatin molecules are electrically neutral , the set time is the shortest . collagen can be partially hydrolyzed by several methods . the type a process is the simplest and most rapid process , in which dilute acid ( e . g . less than 1 m hcl ) is used to partially hydrolyze the collagen . type a processing is generally used with porcine skin and demineralized bovine bone . the type b process uses an alkaline solution to partially hydrolyze the collagen . type b processing is generally used with bovine hide and demineralized bovine bone . finally , enzymes , such as pepsin , may be used to partially hydrolyze collagen . pepsin preferentially cleaves peptide bonds between aromatic amino acids . in collagen , treatment with pepsin converts native collagen , which contains telopeptide , to atelopeptide material , which reduces the level of interchain disulfide bonding that can occur in the collagen tertiary structure . as one example of this method , the gelatin is prepared from the bones of the species into which the compositions are to be implanted , by crushing and defatting the bones followed by demineralization in 0 . 5 n hcl and then soaking for about 24 hours in approximately 300 mg / l pepsin in a 0 . 5 m acetic acid at 33 ° c . we have discovered that the yield of gelatin is enhanced by conducting two such extractions , one at about 30 ° c ., and a second at about 33 ° c ., and then pooling the product . the ph of the resulting solution is brought to about 7 . 0 with sodium hydroxide to denature the pepsin . the temperature of the solution is raised to between about 60 - 65 ° c . for about 12 to 30 minutes and returned to 19 - 20 ° c ., or like temperature at which the gelatin remains soluble , to effect denaturation of remaining collagen and complete conversion to gelatin . the resulting solution is filtered to remove particulates and dialyzed or diafiltered against distilled water for 48 hours in a 30k - 100k molecular weight cut - off ( 30k - 100k mwco ) dialysis or diafiltration membrane . in the improvement of this invention , the gelatin thus produced is next lyophilized , preferably in sealable vials . the vials are filled ( i . e . the vacuum is replaced ) with a dry , inert gas , such as nitrogen , argon or the like , sealed , and then autoclaved ( or otherwise heated , for example in a dry oven , to about 121 - 130 ° c . ), in the sealed environment . this treatment has been found to increase the kinematic viscosity of the gelatin upon subsequent solubilization , permitting a lower effective concentration of the gelatin to be used in the bone paste to achieve gellation at about thirty - eight degrees centigrade , than has heretofore been possible . depending on the level of moisture permitted to remain in the sealed environment , the level of increase in kinematic viscosity , measured subsequently , can be controlled in a dose - dependent fashion ( the increase in viscosity is quenched by humidity ). after lyophilization and thermal treatment , the gelatin is redissolved in phosphate buffered saline ( pbs ) or water to a sufficiently high effective concentration such that a final composition comprising from about 11 - 19 %, and preferably about 15 - 19 % ( w / w ) gelatin may be produced . the improved result of this process is that the thus - treated gelatin sets - up as a gel at the effective gelatin concentration of about 15 - 19 weight percent , as opposed to 20 - 45 weight percent without such treatment . this distinction is clearly evident from the differences between the kinematic viscosity of gelatin produced as described above , with and without thermal treatment of the lyophilized material . the viscosity is higher and set temperature lower for the thermally treated material for the same concentration of gelatin when subjected to the lyophilization ( or other form of drying and moisture reduction ) and thermal cross - linking , than when such treatment is not conducted . the gelatin may be derived from the same or different species than that into which the composition is to be implanted . for example , human , porcine , bovine , equine , or canine gelatin is derived from collagen sources such as bone , skin , tendons , or cartilage , and may then be mixed with dbm or other osteogenic ( osteoinductive or osteoconductive ) materials . as noted above , the collagen is converted to gelatin via , liming , acidification or by enzymatic extraction , for example by pepsin or like enzymatic treatment , followed by denaturation by heat or other means . the gelatin may be derived from tissue by mastication of the tissue , followed by an extended treatment capable of breaking cross - links in the long collagen chains . in one embodiment , the tissue is ground then soaked for about 24 - 72 hours at between about 2 - 40 ° c . in dilute acid , such as 0 . 1 normal acetic acid . preferably , an enzyme such as pepsin at a sufficiently high concentration is added . pepsin concentrations of between about 10 - 20 , 000 i . u ./ liter , 100 - 2 , 000 i . u / liter , or like concentrations are added to the dilute acid at the start of the treatment , with the period of treatment being adjusted according to the enzyme concentration used . solids are removed from the composition , for example by centrifugation , and the supernatant material in solution having a molecular weight of about 50 , 000 daltons or higher is retained . this may be achieved by any of a number of methods known in the art including , but not limited to , dialyzing the supernatant in a 50 , 000 dalton molecular weight cut - off membrane against several changes of solution , ultrafiltration against a membrane having a like molecular weight cut - off , ( mwco ) or gel permeation chromatography through a medium having a 50 , 000 dalton molecular mass cut - off . it will be recognized by those skilled in the art that the higher the mwco of the gelatin , the lower the yield . accordingly , lower mwco gelatin preparations , down to abut 1000 dalton mwco &# 39 ; s could be used , recognizing that undesirable low molecular weight species might thereby be retained . once again , the inclusion of the lyophilization and thermal cross linking disclosed above increases the yield of higher - molecular weight material . the gelatin solution resulting from the foregoing extraction is preferably denatured , for example by heat - treatment to above about 50 to 65 ° c . the denatured protein is then dried and subjected to the inert - gas , thermal cross - linking step described above . thereafter , the gelatin may be stored in a dry state , or reconstituted with physiologically acceptable solutions and stored in a frozen state or it may be freeze - dried after reconstitution or it may be precipitated , for example in a volatile organic solvent , and reconstituted in a solution , such as an isotonic saline solution , at a concentration of between about 15 - 19 % ( w / w ) gelatin . the demineralized bone is preferably in a powdered form , and is preferably composed of particles in the size range between about 80 - 850 μm in diameter . methods for producing demineralized bone powder are known in the art ( see for example u . s . pat . no . 5 , 405 , 390 , herein incorporated by reference for this purpose ), and are not , therefore , elaborated here . demineralized bone powder , extracted by standard techniques , is mixed with the gelatin solution prepared as described above , to form a composition comprising about 0 - 40 % ( w / w ) demineralized bone powder . where present , bone morphogenetic proteins ( bmp ) reduce the percentage of dbm required in the composition . the bmp is preferably present at a concentration of between about 0 . 0001 to 10 mg / ml , 0 . 001 mg / ml to 4 mg / ml , or like concentration , depending on the amount of dbm present ( 0 - 40 % w / w ). in certain embodiments of this invention , and for particular orthopaedic applications in which strength of the bond formed by the thermally sterilized bone paste is important , addition of a bioactive glass is preferred . when added , the bioactive glass lowers the adhesiveness of the composition , but increases the stiffness of the composition upon setting . accordingly , a bioactive glass , such as bioglass ® having a diameter of between about 0 . 5 - 710 μm , is added to the gel / demineralized bone composition . in addition , a composition comprising between about 0 - 40 % ( w / w ) of bioactive glass with the gelatin forming about 11 - 19 % ( w / w ) of the composition is also contemplated . compositions prepared as described above are easily extruded from a syringe , particularly when the temperature is elevated to above about 40 ° c ., for example by immersion in a water bath , by limited treatment in a microwave , by placement in a syringe warmer , or any of a number of other methods for heating the container . the extruded gel is resilient , sticky and easily formable into any desired shape . in addition , the composition retains its strength and is poorly soluble in saline once it sets - up . accordingly , having generally described the composition of this invention , and taking into account the specifics of the exemplary support provided below , the following guidelines for the preparation and use of the composition of this invention are provided : the gelatin from dbm should be prepared at a temperature between about 30 and 37 ° c . while the yield is higher ( 60 %) at 37 ° c ., the quality , based on measured kinematic viscosity , is slightly lower than that produced at 30 ° c . preferably , the gelatin is produced by limited exposure of collagen to an enzyme , such as pepsin , or like enzyme . a concentration of pepsin set at 300 u / l - 500 u / l works well , but those skilled in the art will recognize that a wide range of enzyme concentrations could be tested , based on what is disclosed herein . those skilled in the art will recognize that acid or alkaline processing of skin and tendon may be an alternative to the pepsin technique . the final composition preferably comprises gelatin solution having a viscosity of about 3600 centipoise at 44 ° c . ( when measured in the linear range of a viscosity / sheer rate plot - 0 . 87 / s ), or a kinematic viscosity of about 0 . 7 centistokes at 44 ° c . the concentration of the gelatin in the carrier phase ( i . e . absent added osteogenic components ) is preferably about 15 - 19 % ( w / w ), to ensure that gellation at 38 ° c . will occur in a reasonable amount of time . naturally , those skilled in the art will recognize that , depending on the species of the organism into which the composition is to be implanted , different temperatures may be required . these needs are accommodated by altering the gelatin concentration , increasing the concentration if a higher gel temperature is desired , and lowering the concentration if a lower gel temperature is desired . the dbm content of the composition is defined herein by the concentration required to obtain bone formation similar to that seen with dbm alone . we have found that about 5 - 40 % ( w / w ) dbm in the composition is effective . anything lower than about 5 % seems to do very little by way of bone formation , unless added bmps ( component iii ) are present in the composition , in which case the dbm concentration may be substantially reduced or eliminated altogether . naturally , based on this disclosure , those skilled in the art will recognize that by addition of different concentrations and compositions of bone morphogenetic proteins or other osteogenic or osteoinductive factors , the weight percent of dbm in the composition may be manipulated up or down . in addition , it will be recognized that , depending on the species into which the composition is implanted , the dbm weight percent may need to be adjusted up or down . we have found in in vivo studies that the compositions with dbm contents from 15 to 33 % all produce calcified tissue . we have found that there is a good correlation between the amount of dbm in the composition and the level of bone induction , as long as the dbm concentration is greater than about 19 % ( w / w ). about 38 - 40 % ( w / w ) is the upper mass limit for dbm . accordingly , 0 - 40 % ( w / w ) dbm , and more preferably 5 - 30 % ( w / w ), 7 - 33 %( w / w ) or 15 - 25 % ( w / w ) is desirable for this component . we have observed histologically that , subsequent to implantation into an animal , the gelatin phase is totally absorbed within about 2 weeks . additionally , cartilage and mineralized bone formed within two weeks , with mature bone being evident by about the fourth week . the animals in these studies did not exhibit any gross health problems or any indications of irritation , hematoma , soreness , fever , or weight loss during the study . the composition according to this invention , whether it comprises gelatin and osteogenic components ( i - iv ) may act as a carrier for cortical , cancellous or cortical and cancellous bone chips . such compositions are useful for filling larger bone voids . in addition , when these bone chips are not demineralized , they provide an added spectrum of biological properties not exhibited by the gelatin alone or the gelatin plus osteogenic components ( i - iv ). when present , it is preferred for such bone chips to be in the size range of about 80 μm to about 10 mm . in a further embodiment of this invention , the composition of gelatin and osteogenic components ( i - iv ) is injection molded , vacuum molded , rotation molded , blow molded , extruded or otherwise formed into a solid form . such forms would desirably take the form of vertebral disks , acetabular hemispheres , formable inserts for repairing acetabular cup defects , tubes , ellipsoid shapes for void filling , and intramedullary plugs , which are useful to plug the intramedullary canal of various bones ( i . e . the marrow containing portion of the bone ) to prevent bone cement from entering healthy bone tissue . these forms are produced , for example , by raising the temperature of the composition above its liquefaction temperature ( e . g . about 45 ° c . ), and allowing the composition to gel in a mold of appropriate shape . for such forms , the gelatin content is preferably made as high as possible to ensure that the form remains solid upon grafting into a vertebrate recipient . having generally described the invention , the following examples are provided to show specific features and applications of the invention . it should be recognized that this invention is in no way limited to the specifics of the examples as set forth below , and that the limits of this invention are defined by the claims which are appended hereto . procedure for definition of the set temperature of the thermally sterilized bone paste : in this procedure , the set temperature of the thermally sterilized bone paste is defined . an aliquot of the matrix stored in a frozen state was thawed at approximately 45 ° c . and then drawn into a syringe . the aliquot was then syringed into a tube which is equilibrated at 38 ° c . for 15 minutes . another aliquot was syringed into a vial of distilled water equilibrated at 38 ° c . and allowed to sit for 15 minutes . after this period of time , both aliquots of the matrix were solid and there was little or no dissolution of the matrix into the distilled water . method for defining the concentration of gelatin in the thermally sterilized bone paste : gelatin ( g ) dbm ( g ) water ( g ) 0 . 15 0 . 33 0 . 52 0 . 17 0 . 33 0 . 50 0 . 19 0 . 33 0 . 48 these masses , in a powder form were placed in a plastic bag or like malleable but liquid impermeable container prior to addition of the indicated mass of water . the bag was sealed and shaken to mix the powders thoroughly . the water was added and the bag resealed . the ingredients were kneaded while the bag was submerged in a water bath set at approximately 42 - 47 ° c ., and then the composition was tested for set - up at 38 ° c . as described in example 1 . comparison of the osteoinductivity of grafton ®, ( dbm in glycerol , osteotech ), dbm , and thermally sterilized bone paste with suspended dbm in an athymic rat intramuscular model : known masses of commercially available grafton ® ( dbm in glycerol , osteotech ), dbm , powdered dbm , and the thermally sterilized bone paste of this invention in which dbm was suspended were implanted intramuscularly into athymic nude rats according to the model of strates and urist ( urist , clin . ortho . rel . res . 71 : 271 - 278 , 1970 ). the osteoinductivity of the dbm included in the thermally sterilized bone paste of this invention and that included in grafton ® were found to be identical in a standard osteosarcoma induction assay . after 21 days , explants were removed and analyzed by x - ray and atomic absorption . this analysis revealed a calcium deposition yield of : 0 . 40 ± 0 . 17 g / ml implant for the thermally sterilized bone paste / dbm composition ; 0 . 039 ± 0 . 094 g / ml for grafton ®; and 0 . 15 ± 0 . 072 g / ml for dbm alone . therefore , dbm in the composition of this invention yielded a 2 . 7 fold increase in bone induction than dbm alone , and 10 . 3 fold the bone induction attributable to grafton ®. all of these differences were statistically significant . in addition , the osteoinductive effect of grafton ® was variable , forming bone in only six of ten implants . the dbm / thermally sterilized bone paste of this invention induced bone formation in all implants . thermally sterilized bone paste production , kinematic viscosity , and critical concentration for gellation at 38 ° c . : the kinematic viscosity of porcine gelatin subjected to different heat sterilization treatments ( 121 ° c ., 6 hours ; 130 ° c ., 3 hours ; 150 ° c ., 2 . 5 hours ) was compared to the kinematic viscosity of porcine gelatin which had not been subjected to heat sterilization . the lyophilized gelatin samples treated for 3 hours at 130 ° c . required approximately 40 - 50 minutes to dissolve in sterile water at 55 ° c ., while the lyophilized gelatin samples treated for 6 hours at 121 ° c . required only about 10 minutes to dissolve in sterile water . the porcine material treated for 2 . 5 hours at 150 ° c . was not soluble . we found that the kinematic viscosity of the thermally treated porcine samples ( 121 ° c . and 130 ° c .) increased , as compared with that of the untreated porcine samples . a similar study of human material ( 121 ° c ., 6 hours ), substantially reproduced this result . by way of background , the thermally sterilized bone paste was produced from gelatin extracted from demineralized human cortical bone powder in the size range of 250 - 850 μm , also referred to as demineralized bone matrix powder ( dbm ), by treatment with 0 . 5 m . acetic acid , and pepsin . the dbm was incubated for from 5 to 24 hours at 30 ° c . the supernatant was retained and the solid material was treated with a fresh solution of acetic acid / pepsin at 33 ° c . for another 5 to 24 hours . the supernatants were combined , and the ph was adjusted to 7 . 0 with 1 n naoh , deactivating the pepsin . the solution was pumped at a controlled rate through a tube submerged in a 60 ° c . water bath such that all portions of the solution were subjected to the 60 ° c . treatment for a full 15 minutes , then quenched in ice water . the solution was centrifuged and the supernatant was either dialyzed or diafiltered against a 30 , 000 daltons molecular weight cut off membrane . the retentate was then lyophilized . as a control , dry samples were retained without further heat treatment . the remainder of the material was autoclaved in sealed vials that were first evacuated to 100 millitorr and then back - filled with nitrogen , and sealed . the autoclaving was continued for either six hours at 121 ° c ., or the samples were treated for three hours at 130 ° c . in fig3 the results of heat treating four samples of porcine material at 121 ° c . for 6 hours ( solid circles ), four samples of porcine material at 130 ° c . for 3 hours ( open circles ), is compared with two samples of untreated porcine material ( solid triangles ). in addition , one sample of human material treated for 6 hours at 121 ° c . ( closed square ) is compared with one sample of untreated human material ( closed diamond ), and a sample of human material treated for six hours at 121 ° c ., but under an atmosphere of moist ( about 1 % humidity ) nitrogen ( open diamond ). the kinematic viscosities of dilute concentrations of the thus treated materials ( 0 . 5 %, 0 . 25 %, 0 . 125 %, 0 . 0625 % in phosphate buffered saline solutions ( ph 7 . 4 at 25 ° c . ), were measured with an ubbelhode viscometer at 44 ° c . the kinematic viscosities ( centistokes ) were graphed versus concentration , fig3 . the linear regression was extrapolated to zero to determine the kinematic viscosity at zero concentration . to determine the set temperatures for various thermally sterilized bone paste compositions , gelatin concentrations were varied from 15 w / w % of total composite to 19 w / w % of total composite in water . all thermally sterilized bone paste composites tested contained dbm at a concentration of 33 w / w % of the total composite . different ambient temperatures were used to test whether the thermally sterilized bone paste was solid or liquid , 45 ° c ., 43 ° c ., 41 ° c ., 40 ° c ., 38 ° c ., and 35 . 5 ° c . the set temperature was determined both by subsequent lowering of the ambient temperature and raising of the ambient temperature . the critical concentration of gelatin in a thermally sterilized bone paste composite that was solid at slightly above human body temperature , 38 ° c . to 39 ° c ., was 15 w / w %- 19 w / w % of the total composite for human gelatin , processed at 33 ° c ., and with 33 w / w % of the composite being dbm , the remainder being pbs . the human gelatin processed at 33 ° c . had a zero concentration kinematic viscosity of 0 . 65 centistokes . human gelatin solutions of lower kinematic viscosities were found to have critical concentrations in excess of about 19 w / w %. correspondingly , gelatins with viscosities higher than about 0 . 65 centistokes are expected to thermally cross - link at concentrations lower than about 15 % ( w / w ). procedure for the production of the thermally sterilized bone paste of this invention : this example provides one procedure for the manufacture of bone paste from gelatin and demineralized bone . as fractions of the total mass of composition desired , the following components are weighed ( percentages given are of total composite weight ): dry demineralized bone : 0 - 40 % ( w / w ) lyophilized thermally cross - linkable gelatin : 11 - 19 % ( w / w ) bioglass ®: 0 - 40 % ( w / w ) bone morphogenetic protein : 0 - 10 mg / ml these components are thoroughly blended while dry , and the balance of the composition mass is made up by addition of water , phosphate buffered saline , or any other physiologically acceptable liquid carrier . the composition may be packaged in this form or lyophilized for later reconstruction with water . the malleable properties of the composition are achieved by heating the composition to a temperature sufficient to exceed the liquefaction point of the gelatin , and then allowing the composition to cool to the temperature at which it gels . bloebaum , r . d . human bone ingrowth and materials ; 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spivak , j . ; zhang , z . h . ; prewett , a . b . spine 1993 , 18 , 1634 - 1639 . sperling , l . h . introduction to physical polymer science ; john wiley and sons , inc . : new york , 1992 . mcdonald , t . o . ; britton , b . ; borgmann , a . r . ; robb , c . a . toxicology 1977 , 7 , 37 - 44 . culling , c . f . a . ; allison , r . t . ; barr , w . t . cellular pathology technique ; 4 ed . ; butterworths : london , 1985 .