Patent Application: US-15254805-A

Abstract:
a 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 gelatin matrix . 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 -.

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 - linkable , and the osteogenic components are selected from : ( i ) demineralized bone , preferably derived from the species into which the bone paste is to be implanted ; or ( ii ) bioactive glass ceramic , bioglass ®, bioactive ceramic , calcium phosphate ceramic , hydroxyapatite , hydroxyapatite carbonate , corraline hydroxyapatite , calcined bone , tricalcium phosphate , like material , or mixtures thereof ; or ( iii ) bone morphogenetic protein , tgf - beta , pdgf , or mixtures thereof , natural or recombinant ; or ( iv ) mixtures of ( i )-( iii ). 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 gelation of a solution containing these molecules . the term “ substantially bioabsorbable ” is used herein to describe the property of a material able to cooperate in and become incorporated with new bone formation . 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 , bioactive glass or like ceramics , 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 . pepsin also acts as an esterase , but amides of amino acids are not hydrolyzed . 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 soaking for about 24 hours in approximately 300 mg / l pepsin in a 0 . 5 m acetic acid at 33 ° c . the ph of the resulting solution is brought to 9 . 0 with sodium hydroxide to denature the pepsin , then it is returned to 7 . 0 with hydrochloric acid . the temperature of the solution is raised to 60 ° c . for about 15 to 30 minutes and returned to 4 ° c . to effect denaturation of remaining collagen and complete conversion to gelatin . the resulting solution is filtered to remove particulates and dialyzed against distilled water for 48 hours in a 50k - 100k molecular weight cut - off ( 50k - 100k mwco ) dialysis membrane . after lyophilization , the gelatin is redissolved in phosphate buffered saline ( pbs ) or water to an effective concentration of about 30 - 45 weight percent of gelatin in solution . the gelatin content of the composition is desirably between about 20 - 45 % ( w / w ). 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 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 . the gelatin solution resulting from the foregoing extraction is preferably denatured , for example by heat - treatment to above about 50 ° c . the denatured protein is then stored in a frozen state or it may be freeze - dried or 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 30 - 45 % ( 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 0 . 1 mg / ml , 0 . 001 mg / ml to 0 . 01 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 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 20 - 45 % ( 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 hydrolysis of collagen with the assistance of 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 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 30 - 45 % ( w / w ), ( approximately 50 - 60 % w / v ), to ensure that gelation 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 , 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 . those skilled in the art will recognize the many orthopedic applications of the bone paste of this invention . however , by way of illustration rather than limitation , for purposes of arthrodesis of the spine , one particularly preferred mode of using this composition would be at an early stage of vertebral disk degeneration or subsequent to trauma . diagnosis of trauma or degeneration is followed by formation of a small orifice , or a plurality of small orifices in the intervertebral cartilage at the site of degeneration . the bone paste is then injected into the intervertebral space to induce arthrodesis . a similar procedure could be used with other joints or bone damage . 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 . gelatin production kinematic viscosity and critical concentration for gelation at 38 ° c . in this experiment , the source of collagen was from demineralized human cortical bone powder in the size range of 250 - 850 μm . the demineralized bone matrix powder ( dbm ), 0 . 5 m . acetic acid solution , and pepsin were added to a centrifuge tube . the centrifuge tube was tumbled for 24 hours at the desired temperature : 4 ° c ., 30 ° c ., 33 ° c . or 37 ° c . the ph was adjusted to 9 . 0 then down to 7 . 0 with 1 n naoh and 1n hcl , respectively , deactivating the pepsin . the solution was placed in a 60 ° c . water bath for 15 minutes , then quenched in ice water . the solution was centrifuged and the supernatant was poured into dialysis membrane tubing with a 1000 daltons molecular weight cut off . the supernatant was dialyzed to obtain a 1000 : 1 dilution factor , frozen and lyophilized until completely dry . this experiment was performed in quintuplicates for each temperature . the kinematic viscosities of dilute concentrations of gelatin , 0 . 0625 w / v %, 0 . 125 w / v %, 0 . 25 w / v %, and 0 . 5 w / v % in phosphate buffered saline solutions ( ph 7 . 4 at 25 ° c . ), were measured with an ubbelhode viscometer at 44 ° c . the kinematic viscosities of human gelatin processed at 4 ° c ., 30 ° c ., 33 ° c ., and 37 ° c ., were measured in duplicates , except for 33 ° c . which was only measured once . the kinematic viscosities ( centistokes ) were graphed versus concentration of human gelatin solution , fig3 . the linear regression was extrapolated to zero to determine the kinematic viscosity at zero concentration . the optimum processing temperature was determined by the temperature that yielded the highest solution viscosity at zero concentration , largest slope of the linear regression , greatest yield , and lastly , the gelatin that produced a solid bone composite at slightly above human body temperature . as the processing temperature increased , the yield of gelatin , normalized for the same pepsin to dbm ratio ( 0 . 03 % ( w / v ) pepsin / 1 g dbm ), increased . the kinematic viscosity at zero concentration , or y - intercept , followed a reverse trend . as the processing temperatures increased , the extrapolated kinematic viscosities decreased , table 1 . the human gelatin processed at 30 ° c . had the highest slope on the kinematic viscosity versus concentration plot , 0 . 40 ( centistokes /%), followed by the human gelatin processed at 4 ° c ., 0 . 26 ( centistokes /%), the human gelatin processed at 33 ° c ., 0 . 21 ( centistokes /%), and lastly the human gelatin processed at 37 ° c ., 0 . 17 ( centistokes /%), table 1 . in order to correlate the kinematic viscosities to molecular weight of gelatin , the kinematic viscosities must be translated into intrinsic viscosities . however , the intrinsic viscosities were undefined due to the polyelectrolytic nature of gelatin . as a result , a direct relationship between viscosity and molecular weight of human gelatin can not be made . the set temperatures for various bone paste compositions were determined , table 2 . human gelatin made from dbm via pepsin at 33 ° c ., 35 ° c ., and 37 ° c . was used in the bone paste compositions . gelatin concentrations were varied from 19 w / w % of total composite to 25 w / w % of total composite ( corresponding to 40 w / v % to 60 w / v % gelatin in the carrier matrix ) in a ph 7 . 4 phosphate buffered saline solution ( pbs ). all 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 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 . accordingly , the critical concentration of gelatin in a bone paste composite that was solid at slightly above human body temperature , 38 ° c . to 39 ° c ., was 25 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 . 71 centistokes . human gelatin solutions of lower kinematic viscosities were found to have critical concentrations in excess of about 25 w / w %. correspondingly , gelatins with viscosities higher than about 0 . 71 centistokes are expected to thermally cross - link at concentrations lower than about 25 % ( w / w ). this study demonstrates that the bone paste of this invention is osteoinductive . in addition , this study demonstrates particle sizes for the dbm component of the composition which operate well in promoting new bone growth in an animal into which it is implanted . the intramuscular rat model is the standard model for testing the osteoinductivity of demineralized bone and other osteoinductive factors . strates et al . have used this model for many years ( strates ). as noted in example 1 above , we determined that for gelation at 38 ° c ., a gelatin solution concentration of 40 - 60 % w / v ( 30 - 45 % w / w of the solution absent added osteogenic components ) is required . at this concentration , gelatin acting as a carrier matrix thermally cross - links at 38 ° c . within approximately 8 minutes . in this study we addressed the question of how much dbm must be present in this fixed 40 - 60 % gelatin carrier matrix to induce bone formation which favorably compares with positive controls . we compared 4 different compositions of a dbm / gelatin composite with both positive and negative controls in a rat intramuscular model . the femurs , tibiae , and fibulae were harvested from fresh - killed ( within 24 hours , refrigerated at 4 ° c .) sprague - dawley rats . the diaphyses were cut from the bones and the marrow removed from the mid - shaft with a dissecting probe and sterile water wash . mid - shaft segments were then demineralized in 0 . 6 m . hcl for 24 hours at 4 ° c . with the mass ratio of bone to acid maintained at 1 / 10 or lower . the bone segments were lyophilized and then mixed with dry ice and ground in a lab - scale bone mill . dbm powder was sieved and the fraction from 125 - 450 μm was retained . a carrier matrix of 50 % ( w / v ) gelatin was made by heating phosphate buffered saline ( pbs ) to 60 ° c . and then adding powdered porcine gelatin ( sigma , 300 bloom ) and stirring vigorously . carrier matrix was allowed to age for 15 minutes ( to even out the distribution of gelatin in solution ) and then it was allowed to cool to 50 ° c . dbm was added to the gelatin solution at this point in the following amounts : 0 ( negative control ), 15 , 19 , 24 , and 33 % w / w of the total composite . the composite was blended thoroughly by hand mixing . implants were prepared by ejecting a thread of composite onto a petri dish . these threads were cut into short segments ( ˜ 4 mm . ), weighed , and placed into sterile petri dishes . positive controls were prepared by pelletizing dbm mixed with pbs in a centrifuge . to maintain pellet integrity during the hazards of surgery , these pellets were frozen and implanted as such . young sprague - dawley rats ( 200 - 410 g ) were anesthetized with 86 mg / kg ketamine , and 13 mg / kg xylazine administered intramuscularly ( in the thigh ). a parallel - mid - line incision was made from the tip of the sternum to just above the groin . the lateral aspects of the rectus abdominus were accessed by blunt dissection to either side of the animal . three short incisions were made in the muscle on each side and the implants inserted , followed by 1 to 2 stitches with prolenen 3 - 0 suture ( to mark the location and prevent the ejection of the implant mass ). one positive or one negative control as well as two experimental compositions were inserted on each side . implant locations were random except that each rat had one positive control on one side and one negative control on the contralateral side . animals were returned to their cages and provided food and water ad - lib . all members of the study group were kept for 4 weeks except one animal ( r1 ) which was sacrificed after 2 weeks for histology . after 4 weeks , animals were sacrificed with an overdose of nembutal . the rectus abdominus was removed by sharp dissection , removing as much tissue as possible . each muscle was notched to mark the superior side of the animal and placed into a labeled petri dish . the muscle was x - rayed with mammography equipment , using mammography film ( dupont ). roentgenograms were analyzed using a digital camera attached to an apple lcii equipped with nih image 4 . 1 software . images were thresholded to highlight the implant shadow and then the area of the shadow was determined by pixel counting . two of each variety of explant were removed from the muscle and fixed in 10 % buffered formalin . histological sections were taken and consecutive sections were stained with h & amp ; e and masson &# 39 ; s trichrome stain . these histological samples were examined by a qualified pathologist . remaining explants were cut from the muscle tissue and ashed in a muffle furnace for 4 . 5 hours at 700 - 750 ° c . ash weight was determined and normalized to original implant weight . ash was dissolved in 1 . 0n hcl and analyzed for calcium content by atomic absorption spectroscopy . all analyses were conducted in a blinded manner with decoding done only after processing of the data was complete . two week histology samples of 15 % and 19 % dbm composites indicated that bone formation was occurring , even at this early date . the route of bone formation is not readily apparent , but appears to be endochondral . four week histology samples revealed that mature bone was formed at the site of implantation . the quality of bone formed was comparable to that of natural bone as shown by the ash and percent calcium analyses . all implants containing dbm were found to lead to the production of some bone . those containing greater than about 20 % dbm yielded the highest quality bone . fig4 a and 4b provide photomicrographs of sections of implants after four weeks in vivo in the rat intramuscular model . we found that 33 % ( w / w ) dbm in gelatin carrier ( fig4 b ) according to this invention produced as much new bone as pure , 100 % dbm ( fig4 a ). in these figures , the following structures are evident : 10 is mature bone , as evidenced by red stain uptake from masson &# 39 ; s stain ; 20 is new cartilage formation , as evidenced by uptake of blue stain from masson &# 39 ; s stain and the presence of cells ; 30 is residual dbm , as evidenced by uptake of blue stain and the absence of cells , from which all cartilagenous and bone structures in the muscle cross section arose ; and 40 is immature bone , as evidenced by light blue staining and the presence of cells . the cells seen are osteoclasts , degrading the newly formed cartilage , and osteoblasts , laying down new bone . in addition , vascular infiltration in the mature bone is evident in the masson &# 39 ; s stained sections , from which the black and white prints were made . there was no statistically significant difference , using a 2σ test , in ash content between the negative control , the positive control , or compositions comprising 15 % or 19 % ( w / w ) dbm . this does not necessarily imply that these compositions do not work ( examination of the roentgenograms obviates this conclusion ). rather , it indicates that the sensitivity of the ash method does not allow the detection of the difference . examination of the data for the 24 % and 33 % composites indicates that they are significantly better than 19 %, 15 %, and the negative controls , and are not significantly different from the ( positive ) control , see table 3 : the atomic absorption spectroscopy of ashed compositions of dbm / gelatin composites yielded the amount of calcium in the samples . the 15 % and 19 % compositions did not show a statistically significant difference from the negative controls . however , it is expected that with greater assay sensitivity , positive effects of dbm at concentrations as low as about 7 % ( w / w ) in gelatin carrier would be measurable . the average calcium content produced by compositions greater than or equal to 24 % appeared to be proportional to the amount of dbm , by weight , in the composition : gross examination / comparison of the x - rays reveals that the 24 % and 33 % compositions are not significantly different from the (+) controls . the 15 % and 19 % compositions do not appear to generate significant bone . however , it is expected that with greater assay sensitivity , positive effects of dbm at concentrations as low as about 7 % ( w / w ) in gelatin carrier would be measurable . no bone formation was apparent on the x - rays at the locations of the (−) controls . accordingly , we conclude that dbm at a concentration of between about 24 % to 33 % ( w / w ) in gelatin is active in inducing bone formation . these same data indicate that concentrations of dbm below about 20 % are less effective in generating significant bone in comparison to positive controls . it is noted that grafton ™ contains only 8 % dbm in a glycerol carrier . 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 : 20 - 45 % ( w / w ) bioglass ®: 0 - 40 % ( w / w ) bone morphogenetic protein : 0 . 001 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 mateaks ; bloebaum , r . d ., ed . ; society for biomaterials : denver , colo ., 1996 . einhorn , t . a . enhancement of bone repair using biomaterials ; einhorn , t . a ., ed . ; society for biomaterials : denver , colo ., 1996 . benedict , j . j . the role of carrier matrices on bone induction in vivo ; benedict , j . j ., ed . ; society for biomaterials : denver , colo ., 1996 . strates , b . ; tiedeman , j . european journal of experimental musculoskeletal research 1993 , 2 , 61 - 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