Puros® Demineralized Bone Matrix (DBM) Research Anthology

Puros® Demineralized Bone Matrix (DBM) Research Anthology Table of Contents Puros Demineralized Bone Matrix (DBM) Research Anthology Puros Demineralized Bone Matrix (DBM).............. 1 Osteoinductivity of Puros DBM Putty in Athymic Rat Model............................................ 7 Effect of Terminal Gamma Sterilization on Osteoinductivity..............................................10 Comparative Handling Properties of Puros Demineralized Bone Matrix Putty................12 Puros Demineralized Bone Matrix Patient Safety Through Redundant Safeguards.............................14 Osteoinductivity of Puros DBM Putty in Athymic Rat Model Following a One Year Shelf-Life Study.................................................... 15 Zimmer ZimmerTechnical Technicalmm emo emo Zimmer Technical memo Zimmer Technical memo Puros® Demineralized Bone Matrix (DBM) Puros Puros®®®®Demineralized Demineralized DemineralizedBone Bone BoneMatrix Matrix Matrix(DBM) (DBM) (DBM) Zimmer Technical Memo Puros Puros Demineralized Bone Matrix (DBM) Overview OverviewofofPuros PurosDemineralized DemineralizedBone BoneMatrix Matrix Overview ofPuros Puros Demineralized Bone Matrix Overview of Demineralized Bone Matrix Overview of Puros Demineralized Bone Matrix Puros PurosDemineralized DemineralizedBone BoneMatrix Matrix(DBM) (DBM)represents representsadvancement advancementininbone bonegraft graftsubstitute substitutetechnology technologyutilizing utilizing Puros Demineralized Bone Matrix (DBM) represents advancement in bone graft substitute technology utilizing demineralized demineralized bone bonematrix matrix(DBM) (DBM) inina aputty putty formulation. formulation. It Itisis100% 100% demineralized human human bone; bone;ininaddition, addition, putty puttywith with Puros Demineralized Bone Matrix (DBM) represents advancement indemineralized bone graft substitute technology utilizing demineralized bone matrix (DBM) in a putty formulation. It is 100% demineralized human bone; in addition, putty with chips mineralized cancellous chips. allograft DBM bone graft substitutes chipscontains containsbone mineralized corticalin cancellous chips.Many Many allograft DBMbased based bone graftin substitutes currently demineralized matrix cortical (DBM) a putty formulation. Itother isother 100% demineralized human bone; addition,currently putty with chips contains mineralized cortical cancellous chips. Many other allograft DBM based bone graft substitutes currently 1 1 available available contain contain a anon-DBM-based non-DBM-based carrier carriertotochips. facilitate facilitate handling handling and andgraft graft containment. containment. Puros Puros DBM DBM Putty Puttydiffers differs chips contains mineralized cortical cancellous Many other allograft DBM based bone graft substitutes currently available contain a non-DBM-based carrier to facilitate handling and graft containment.11 Puros DBM Putty differs from fromthese these products products because becauseitsitscarrier carrier isisalso also DBMfrom from the thesame same donor, donor, froma adifferent different stage stage ofofthe the proprietary proprietary available contain a non-DBM-based carrier to DBM facilitate handling and graft from containment. Puros DBM Putty differs from these products because its carrier is also DBM from the same donor, from a different stage of the proprietary processing processing methodology. methodology. from these products because its carrier is also DBM from the same donor, from a different stage of the proprietary processing methodology. processing methodology. Themixture mixtureofofhuman humanDBM DBMpowder powderwith withthe theDBM DBMcarrier carrier The The mixture of human DBM powder with the DBM carrier from thesame same donorresults resultsinpowder ina aPuros Puros DBM product with from the DBM with The mixture ofdonor human DBM with the product DBM carrier from the same donor results in a Puros DBM product with putty-like consistency exhibiting excellent handling a aputty-like consistency exhibiting excellent from the same donor results in a Puros DBMhandling product with a putty-like consistency exhibiting excellent handling propertiesand andgraft graftcontainment. containment. Thereisisalso also Puros a aPuros aproperties putty-like consistency exhibitingThere excellent handling properties and graft containment. There is also a Puros DBMPutty Putty formulation thatcontains contains cortico-cancellous DBM formulation that cortico-cancellous properties and graft containment. There is also a Puros DBM Putty formulation that contains cortico-cancellous chipsfrom fromthe thesame samedonor donor asthat thatofofthe theDBM. DBM.The The chips DBM Putty formulation thatascontains cortico-cancellous chips from the same donor as that of the DBM. The product easy-to-use andconvenient ready product isiseasy-to-use and asas it itisisready chips from the same donor asconvenient that of the DBM. The forfor product is easy-to-use and convenient as it is ready for immediate usewithout without extra preparation required inthe the immediate use extra preparation required product is easy-to-use and convenient as it is readyinfor immediate use without extra preparation required in the OR.It Itshould should bestored stored controlled room temperature OR. atatcontrolled room temperature immediate usebe without extra preparation required in the OR. It should be stored at controlled room temperature (15°-25°C). (15°-25°C). OR. It should be stored at controlled room temperature (15°-25°C). (15°-25°C). Boththe theactive activeDBM DBMpowder powderand andthe thecarrier carrierare are100% 100%dedeBoth Both the active DBM powder and the carrier are 100% demineralized human bone from thethe same donor. PurosDBM DBM mineralized human bone from the same donor. Both the active DBM powder and carrier arePuros 100% demineralized human bone from the same donor. Puros DBM consistsofofactive activehuman human DBM mixed with osteoconducconsists DBM mixed osteoconducmineralized human bone from the samewith donor. Puros DBM consists of active human DBM mixed with osteoconductiveDBM DBM derived fromthe thesame same donor. The onlyexcipiexcipitive from donor. The only consists of derived active human DBM mixed with osteoconductive DBM derived from the same donor. The only excipientin inthe theproduct productisfrom issterile sterile water. Because the product ent Because the product isis tive DBM derived thewater. same donor. The only excipient in the product is sterile water. Because the product is 100% natural bone matrix,water. resorbable and remodels 100% natural bone it itisisresorbable remodels ent in the product ismatrix, sterile Because and the product is 100% natural bone matrix, it is resorbable and remodels readily. Through proprietary procedures, Puros readily. Through proprietary procedures, Puros 100% natural bone matrix, it processing isprocessing resorbable and remodels readily. Through proprietary processing procedures, Puros DBMPutty Puttyisisgenerated generated such thatit itretains retains highdegree degree DBM such that a ahigh readily. Through proprietary processing procedures, Puros DBM Putty is generated such that it retains a high degree osteoinductive potential* inaddition addition ofofosteoinductive potential* totoserving asasanan DBM Putty is generated suchinthat it retains aserving high degree of osteoinductive potential* in addition to serving as an osteoconductive matrix.Puros Puros DBMPutty Putty pre-mixed osteoconductive DBM isispre-mixed of osteoinductivematrix. potential* in addition to serving as an osteoconductive matrix. Puros DBM Putty is pre-mixed anddelivered deliveredininopen openbore bore dispensers andjars serveasasa a and dispensers and totoserve osteoconductive matrix. Puros DBM Putty isjars pre-mixed and delivered in open bore dispensers and jars to serve as a ‘ready-to-use’ product. ‘ready-to-use’ product. and delivered in open bore dispensers and jars to serve as a ‘ready-to-use’ product. ‘ready-to-use’ product. additiontotoextensive extensivedonor donorscreening, screening,procedures procedures InInaddition In addition to extensive donor screening, procedures properhandling thetissue, tissue, andRTI’s RTI’s proprietary forfor proper ofofthe and proprietary In addition tohandling extensive donor screening, procedures for proper handling of the tissue, and RTI’s proprietary processing methods, finalproduct product terminally sterilprocessing methods, isisterminally sterilfor proper handling ofthe thefinal tissue, and RTI’s proprietary processing methods, the final product is terminally sterilizedusing usinglow-temperature, low-temperature, low-dose gamma irradiation ized irradiation processing methods, the finallow-dose product isgamma terminally steril- toto ized using low-temperature, low-dose gamma irradiation to provide safety againstdisease disease transmission. This methodolprovide safety against transmission. This methodolized using low-temperature, low-dose gamma irradiation to provide safety against™ ™ disease transmission. This methodology,the the Cancelle ogy, Cancelle SPSP DBM DBM Sterilization Sterilization process, process, has has been been provide safety against disease transmission. This methodol™ ogy, the Cancelle SP ™ DBM Sterilization process, has been validated validated totoinactivate inactivate orremove remove bacteria, bacteria, viruses, viruses, fungi fungi ogy, the Cancelle SP or DBM Sterilization process, has been validated to inactivate or remove bacteria, viruses, fungi and andspores, spores, whilepreserving preserving protein protein activity. activity. validated to while inactivate or remove bacteria, viruses, fungi and spores, while preserving protein activity. and spores, while preserving protein activity. History HistoryofofDBM DBM History of DBM History of DBM History of DBM Bone Boneconsists consists ofoftwo twomajor majorcomponents: components:organic organicproteins proteins Bone consists of two major components: organic proteins and andinorganic inorganicmineral. Collagen Collagen isisthe themain main constituent constituent ofof Bone consists ofmineral. two major components: organic proteins and inorganic mineral. Collagen is the main constituent of the theorganic organicmaterial material that that gives givesbone bone itsits toughness toughness and and of and inorganic mineral. Collagen is the main constituent the organic material that gives bone its toughness and resilience. resilience. Inaddition, addition, there are are many many growthfactor factor the organicIn material thatthere gives bone itsgrowth toughness and resilience. In addition, there are many growth factor proteins proteinsthat that closelyregulate regulate bone bone formation formation and resilience. In closely addition, there are many growthand factor proteins that2,closely regulate bone formation and 32, 3 remodeling. remodeling. Proteins Proteins constitute constitute nearly30-35% 30-35% proteins that closely regulate bonenearly formation and ofof remodeling.2,2, 33 Proteins constitute nearly 30-35% of bone bonebybyweight. weight. remodeling. Proteins constitute nearly 30-35% of bone by weight. bone by weight. * DBM * DBM induced induced bone bone formation formation when when implanted implanted in in anan athymic athymic ratrat assay. assay.Findings Findings *from DBM induced bone formation when implanted in an ratclinical assay. Findings from anan animal animal model model areare notnot necessarily necessarily predictive predictive of athymic of human human clinical results. results. *from DBM bone formation when implanted in an rat assay. Findings an induced animal model are not necessarily predictive of athymic human clinical results. from an animal model are not necessarily predictive of human clinical results. The Themineral mineralpart partofofbone, bone,which whichconstitutes constitutesnearly nearly The mineral part of bone, which constitutes nearly 65-70% 65-70% bybyweight, weight, isessentially essentially comprised comprisednearly ofofcalcium calcium The mineral part of is bone, which constitutes 65-70% by weight, is essentially comprised of calcium phosphate phosphate saltsthat that give bone boneitsits stiffness stiffnessand andstrength. strength. 65-70% bysalts weight, isgive essentially comprised of calcium phosphate salts that give bone its stiffness and strength. This Thiscomponent component ofofbone bonedissolves dissolves readily readilyin inacid acid phosphate salts that give bone its stiffness and strength. This component of bone dissolves readily in acid medium medium resulting resulting indemineralized demineralized bone bonematrix matrix rendering This component ofinbone dissolves readily in acidrendering medium resulting in demineralized bone matrix 4, 54, 5 rendering the thegrowth growth factors factors more readily readilyaccessible accessible totoinitiate initiate medium resulting inmore demineralized bone matrix rendering 4, 5 the growth factors more readily accessible4, 5 to initiate thegrowth cascade cascade ofofbone bone regeneration regeneration while whilesimultaneously simultaneously the factors more readily accessible to initiate the cascade of bone regeneration while simultaneously retaining retaining the the natural scaffold scaffoldmatrix. matrix. the cascade ofnatural bone regeneration while simultaneously retaining the natural scaffold matrix. retaining the natural scaffold matrix. The Thefirst firstreported reportedclinical clinicaluse useofofDBM DBMdates datesback backtoto1889 1889 The first reported clinical use of DBM dates back to 1889 6 6 when when Senn Senn used used DBM DBM as as a a vehicle vehicle for for the the delivery delivery of of The first reported clinical use of DBM dates back to 1889 when Senn66 used DBM as a vehicle for the delivery of antiseptics antiseptics inthe thetreatment treatment ofbone bonecavities cavities and and of when Senn inused DBM as aof vehicle for the delivery antiseptics in the treatment of bone cavities and demonstrated demonstrated that that demineralized demineralized bovine bovine bone bone could could antiseptics in the treatment of bone cavities and demonstrated that demineralized bovine bone could successfully successfullyrepair repair large largeosseous osseousbovine defects defects that thatwould wouldnot not demonstrated that demineralized bone could successfully repair large osseous defects that would not completely completelyheal healbyby themselves. themselves. Hedemonstrated demonstrated this this successfully repair large osseousHe defects that would not completely heal by themselves. He demonstrated this using usingDBM DBM ininlong long bone boneand andcranial cranial defects defectsinindogs, dogs, completely heal by themselves. He demonstrated this using DBM in long bone and cranial defects in dogs, and andtibial tibial and and femoral defects defects ininhumans. humans. For For decades using DBM infemoral long bone and cranial defects indecades dogs, and tibial and femoral defects in7humans. For decades 7 this thistibial field fieldlay laydormant dormantuntil untilUrist Urist (in (in1965) 1965) published published and and femoral defects in humans. For decadeshishis this field lay dormant until Urist77 (in 1965) published his seminal seminal work demonstrating demonstrating the theosteoinductive osteoinductive this fieldwork lay dormant until Urist (in 1965) published his seminal work demonstrating the osteoinductive properties properties ofofdemineralized demineralizedbone. bone. HeHeshowed showedthat thatDBM DBM seminal work demonstrating the osteoinductive properties of demineralized bone. He showed that DBM had hadthe thecapacity capacity totoinduce inducebone boneformation formation ininathat anon-bony non-bony properties of demineralized bone. He showed DBM had the capacity to induce bone formation in a non-bony tissue tissue byimplantation implantation ininmuscle muscle pouches pouchesof It Itisisnow now had thebycapacity to induce bone formation inofrats. arats. non-bony tissue by implantation in muscle pouches of rats. It is now known known that that only onlyhighly highly osteoinductive substances substances tissue by implantation inosteoinductive muscle pouches of rats. Itcan iscan now known that only highly osteoinductive substances can induce inducebone bone formation formation atsuch suchheterotopic heterotopic sites. sites.Urist Urist also known that only highly at osteoinductive substances can also induce bone formation at such heterotopic sites. Urist also demonstrated demonstrated that thatdemineralized demineralized bone bonecould could bebeimplanted implanted induce bone formation at such heterotopic sites. Urist also demonstrated that demineralized bone could be implanted into intohuman humanlong long bonedefects defectsand and lumbar lumbar vertebrae vertebrae toto demonstrated thatbone demineralized bone could be implanted into human long bone defects and lumbar vertebrae to regenerate regenerate bone. Further, Further, hehedemonstrated demonstrated that thatthe the to into humanbone. long bone defects and lumbar vertebrae regenerate bone. Further, he demonstrated that the osteoinductive osteoinductive fraction could could bebeextracted extractedfrom from regenerate bone.fraction Further, he demonstrated that the osteoinductive fraction could be extracted from demineralized demineralizedbone. bone.HeHe identified identified this thisfraction fraction bone osteoinductive fraction could be extracted fromasasbone demineralized bone. He8 8identified this fraction as bone morphogenetic morphogenetic protein protein (BMP)after after establishing thatthe the demineralized bone. He8 (BMP) identified thisestablishing fraction as that bone morphogenetic protein8 (BMP) 9 9 after establishing that the activity activityresided resided inina aprotein. protein. BMPs BMPs areknown knowntoto signal signal morphogenetic protein (BMP) after are establishing that the 9 activity resided in a protein.9 BMPs are known to signal precursor precursor cells cellsto bone bone formation. formation. activity resided intoregulate aregulate protein. BMPs are known to signal precursor cells to regulate bone formation. precursor cells to regulate bone formation. Since Since1965, 1965,numerous numerousanimal animaland andclinical clinicalstudies studieshave have Since 1965, numerous animal and clinical studies have demonstrated demonstrated the theeffectiveness effectiveness ofofDBM DBMinin a avariety variety ofof Since 1965, numerous animal and clinical studies have demonstrated the effectiveness of DBM in 10 10a variety of osseous osseousdefects. defects. In1981, 1981,Mulliken Mulliken etetal.al. reported reported using using demonstrated theIneffectiveness of DBM in a variety of 10 osseous defects. In 1981, Mulliken et al.10 reported using DBM DBMinin craniomaxillofacial craniomaxillofacial defects defects in4444patients patientswith with osseous defects. In 1981, Mulliken etinal. reported using DBM in craniomaxillofacial defects in 44 patients with 5555implants implants including includingdemineralized demineralized bone bone powder powderwith and and DBM in craniomaxillofacial defects in 44 patients 55 implants including demineralized bone powder and allograft allograft cancellous cancellous chips chips and andblocks. blocks. Radiographic Radiographic 55 implants including demineralized bone powder and allograft cancellous chips and blocks. Radiographic healing healingwas wasevident evident inin3-6 3-6 months, months, and and biopsy biopsysections sections allograft cancellous chips and blocks. Radiographic healing was evident in 3-6 months, and biopsy sections revealed revealed evidence evidence induced bone bonethroughout throughout thedefects defects healing was evidentofof ininduced 3-6 months, and biopsy the sections revealed evidence of induced bone throughout the defects rather ratherthan than bone boneformation formation atatbone the theedge edge ofofthe thedefect defect asasinin revealed evidence of induced throughout the defects rather than bone formation at the edge of the defect as in creeping creeping substitution. substitution. rather than bone formation at the edge of the defect as in creeping substitution. creeping substitution. ©2009 ©2009 Zimmer Zimmer Inc.Inc. AllAll rights rights reserved reserved ©2009 Zimmer Inc. All rights reserved ©2009 Zimmer Inc. All rights reserved 1 11 Tiedman Tiedman et al. et 11al.studied studied thethe efficacy efficacy of DBM of DBM alone alone andand 11with 11 as a as composite a composite with bone bone marrow marrow forof for several indications indications Tiedman et al.studied studied the efficacy ofseveral DBM alone Tiedman et al. the efficacy DBM alone andand 11with including including spinal fusion, fusion, augmentation augmentation of bone of bone inindications total inand total a composite with bone marrow for several as aas composite bone marrow forof several indications Tiedman etspinal al. studied the efficacy DBM alone joint joint replacement, replacement, acute acute fractures, fractures, non-unions, non-unions, defects, defects, including spinal fusion, augmentation of bone in total including spinal fusion, augmentation of bone in total as a composite with bone marrow for several indications andjoint and joint joint arthrodesis. arthrodesis. Union Union occurred occurred in in 77% ofin the of the cases. cases. replacement, acute fractures, non-unions, defects, joint replacement, acute fractures, non-unions, defects, including spinal fusion, augmentation of77% bone total The author author stated stated that that “patients “patients demonstrate demonstrate the the efficacy efficacy and joint arthrodesis. Union occurred in 77% of the cases. andThe joint arthrodesis. Union occurred in 77% ofdefects, the cases. joint replacement, acute fractures, non-unions, of ofjoint DBM to graft to stated graft osseous osseous defects defects ranging ranging from from acute The author that “patients demonstrate the efficacy TheDBM author stated that “patients demonstrate the efficacy and arthrodesis. Union occurred in 77% ofacute the cases. fractures fractures with with bone bone loss, loss, to fracture to fracture non-unions, non-unions, toacute bone to bone of DBM to graft osseous defects ranging from of DBM to graft osseous defects ranging from The author stated that “patients demonstrate theacute efficacy defects defects resulting resulting from from tumor tumor excision excision or total or total joint joint fractures with bone loss, to fracture non-unions, fractures with bone loss, to fracture non-unions, to bone of DBM to graft osseous defects ranging from acuteto bone revision revision surgery.” surgery.” defects resulting from tumor excision or total joint defects resulting from tumor excision or total joint fractures with bone loss, to fracture non-unions, to bone revision surgery.” revision surgery.” 12 or 12 total joint 13 13 defects resulting from tumor excision More More recently, recently, Leriche Leriche andand Policard Policard , Levander , Levander , , 14 14 15 15 16 16 revision surgery.” 12 12 established 13themselves LaCroix LaCroix , Huggins , Huggins , and , and Reddi Reddi established themselves More recently, Leriche and Policard , Levander More recently, Leriche and Policard , Levander , 13, 14 15 16 14 15 16 12 13 as pioneers as pioneers in the in the field field of induced of induced bone bone formation. formation. LaCroix , Huggins , and Reddiestablished themselves LaCroix , Huggins , and Reddi themselves More recently, Leriche and Policard ,established Levander , 14 in the 15 field 16 bone as pioneers in the of induced bone formation. as pioneers field of induced formation. LaCroix , Huggins , and Reddi established themselves Osteoinductive Osteoinductive Nature Nature of DBM of DBM as pioneers in the field of induced bone formation. Osteoinductive Nature of DBM Osteoinductive Nature of DBM Native Native bone bone is the is the largest largest reservoir reservoir of osteoinductive of osteoinductive Osteoinductive Nature of DBM Osteoinductive of DBM factors. factors. These These factors reside reside in aindormant a of dormant fashion fashion bound bound Native bone isNature the largest reservoir of osteoinductive Native bone is factors the largest reservoir osteoinductive to the to the collagen collagen matrix matrix in reside bone. in bone. Following injury, injury, factors. These factors a of dormant fashion bound factors. These reside in Following aindormant fashion bound Native bone is factors the largest reservoir osteoinductive the the growth growth factors factors areare mobilized mobilized and and activated activated so they so they to the collagen matrix in bone. Following injury, to the collagen matrix in bone. injury, factors. These factors reside in Following a dormant fashion bound can stimulate migration, differentiation and proliferation can stimulate migration, and proliferation the growth factors mobilized activated so they the growth factors areare mobilized andand activated so they to the collagen matrix in differentiation bone. Following injury, of surrounding progenitor cells to initiate the of progenitor cells toand initiate the cascade can stimulate migration, differentiation and proliferation can stimulate migration, differentiation and proliferation thesurrounding growth factors are mobilized activated socascade theyof of 17 17 bone repair bone repair . migration, The . The demineralization demineralization process not not only only of surrounding progenitor cells toprocess initiate the cascade of progenitor cells to initiate theproliferation cascade of of cansurrounding stimulate differentiation and 17 osteoinductive 17 osteoinductive retains retains these these factors factors but but also also helps helps toonly toof bone repair . The demineralization process not bone repair . The demineralization process not only of surrounding progenitor cells to initiate the cascade 17 osteoinductive expose expose them them surrounding to demineralization surrounding cells cells forbut for easy easy access. access. Thus, retains these osteoinductive factors but also helps to retains these factors also helps to Thus, bone repair .toThe process not only DBM DBM can can provide provide an optimal an optimal natural natural matrix matrix for for expose them to surrounding cells for easy access. Thus, expose them to surrounding cells for easy access. Thus, retains these osteoinductive factors but also helps to osteogenic osteogenic progenitor progenitor cells cells tocells adhere, to natural adhere, differentiate differentiate andand DBM can provide an optimal matrix DBM can provide an optimal natural matrix for forThus, expose them to surrounding for easy access. form form bone bone ifprogenitor the ifprogenitor the osteoinductive factors remain remain active. osteogenic cells tofactors adhere, differentiate osteogenic cells to natural adhere, differentiate andand DBM can provide anosteoinductive optimal matrix foractive. Because Because demineralization demineralization of bone results results inremain exposing in active. exposing form bone if the osteoinductive factors active. form bone ifprogenitor the osteoinductive factors remain osteogenic cellsoftobone adhere, differentiate andthethe growth growth factors factors that that are are bound bound to the to the collagenous collagenous matrix, matrix, Because demineralization of bone results in exposing Because demineralization of bone results in exposing thethe form bone if the osteoinductive factors remain active. it is it critical is critical to optimize to optimize the the demineralization demineralization process process to growth factors that are bound to the collagenous matrix, growth factors that are bound to the collagenous matrix, Because demineralization of bone results in exposing theto ensure ensure that growth growth factors remain active active through through thisthis is that critical to optimize the demineralization process it isit critical to optimize theremain demineralization process to to growth factors that arefactors bound to the collagenous matrix, process. The The observed high variability variability inthrough the inthrough the ensure that growth factors remain active ensure that growth factors remain active thisthis it isprocess. critical toobserved optimize thehigh demineralization process to osteoinductive osteoinductive potential potential ofhigh commercially of commercially available available DBMs DBMs process. The observed variability in the process. Thegrowth observed high variability inthrough the ensure that factors remain active this can, can, to a to large a large extent, extent, be attributed be attributed to the to the differences differences in in osteoinductive potential of commercially available DBMs osteoinductive potentialhigh of commercially available DBMs process. The observed variability in the 18 18 the the demineralization demineralization processes processes employed. employed. can, to a large extent, be attributed to the differences in can, to a large extent, be attributed to the differences in osteoinductive potential of commercially available DBMs 18 18 the demineralization processes employed. the demineralization processes employed. can, to a large extent, be attributed to the differences in Osteoinductive Osteoinductive factors factors areare critical critical to bone to bone induction induction 18 thevivo. demineralization processes employed. in in vivo. Successful Successful delivery delivery ofcritical these of these requires requires their their Osteoinductive factors to factors bone induction Osteoinductive factors areare critical to factors bone induction association association with with suitable suitable matrices. These These matrices, matrices, among among in vivo. Successful delivery of these factors requires their in vivo. Successful delivery of these factors requires their Osteoinductive factors arematrices. critical to bone induction other other characteristics, characteristics, should should bethese biocompatible, be biocompatible, association with suitable matrices. These matrices, among association with suitable matrices. These matrices, among in vivo. Successful delivery of factors requires their biodegradable biodegradable and and capable capable ofbemaintaining ofbiocompatible, thethe proteins proteins other characteristics, should bemaintaining biocompatible, other characteristics, should association with suitable matrices. These matrices, among in their in their active active state. state. They They should should serve serve as a as reservoir a reservoir biodegradable and capable of maintaining the biodegradable and capable of maintaining the proteins other characteristics, should be biocompatible, proteins fortheir for these these proteins proteins for for extended extended periods periods of atime. of in their active They serve as atime. reservoir in active state. They should serve as reservoir biodegradable andstate. capable ofshould maintaining the proteins Demineralized Demineralized bone bone provides provides all all these these features features andand is is for these proteins for extended periods ofreservoir time. for these proteins for extended periods of atime. in their active state. They should serve as also also thethe natural natural reservoir reservoir for for the the proteins proteins vivo. in vivo. BMPs Demineralized bone provides all these features and is Demineralized bone provides all these features and isBMPs for these proteins for extended periods ofin time. 19 19 1-16 1-16 have have been been identified identified in DBM. in DBM. These These proteins proteins start also the natural reservoir for the proteins in vivo. BMPs also the naturalbone reservoir for the in vivo.and BMPs Demineralized provides allproteins these features isstart 19 19 These the the osteoinduction osteoinduction cascade cascade by by initiating initiating chemotaxis chemotaxis of of 1-16 have been identified in DBM. proteins 1-16 have been identified in DBM. These proteins start also the natural reservoir for the proteins in vivo. BMPsstart 19 migration progenitor progenitor cells cells which which is the isby the directed directed migration of cells ofstart in in the osteoinduction cascade by initiating chemotaxis of the osteoinduction cascade initiating chemotaxis ofcells 1-16 have been identified in DBM. These proteins response response tocells atochemical a which chemical gradient gradient of signals of migration signals released released from from progenitor cells which is the directed migration of progenitor is the directed of cells in in the osteoinduction cascade by initiating chemotaxis ofcells 20 insoluble insoluble DBM. DBM. response a20 chemical gradient of migration signals released from response to ato chemical gradient of signals released from progenitor cells which is the directed of cells in 20 20 insoluble DBM. insoluble DBM. response to a chemical gradient of signals released from DBM: DBM: AnAn Alternative Alternative to Autograft to Autograft 20 insoluble DBM. DBM: An Alternative to Autograft DBM: An Alternative to Autograft Surgical Surgical management management of aofnumber a number of orthopedic of orthopedic problems problems DBM: An Alternative to Autograft necessitates necessitates thethe useuse of of bone ofaof bone graft toofensure toorthopedic a clinically a clinically Surgical management agraft number ofensure orthopedic problems Surgical management problems DBM: An Alternative tonumber Autograft successful successful outcome. outcome. necessitates the ofabone graft toorthopedic ensure a clinically necessitates the useuse of of bone graft toofensure a clinically Surgical management number problems successful outcome. successful outcome. necessitates the use of bone graft to ensure clinically Bone Bone undergoes undergoes constant constant remodeling remodeling in ainliving aa living successful outcome. organism. organism. Small Small fractures fractures of bone of bone heal heal without without thethe need need Bone undergoes constant remodeling a living Bone undergoes constant remodeling in ainliving for for intervention intervention because because of its of high its high potential potential to regenerate. to regenerate. organism. Small fractures of bone heal without the need organism. Small constant fracturesremodeling of bone healinwithout Bone undergoes a livingthe need for intervention because of its high potential to regenerate. for intervention because of its high potential to regenerate. organism. Small fractures of bone heal without the need for intervention because of its high potential to regenerate. However, larger defects created either trauma However, larger defects created either by by trauma or or surgical procedures not self-heal Fast surgical procedures willwill notcreated self-heal effectively. Fast However, larger defects either by trauma or However, larger defects created either byeffectively. trauma or healing with good quality bone occurs when the defect healing with good quality bone occurs when the defect surgical procedures will not self-heal effectively. Fast surgical will created not self-heal Fast is is However,procedures larger defects eithereffectively. by trauma or filled with proper matrix (osteoconductive agent) that filled with proper matrix (osteoconductive agent) that can healing with good quality bone occurs when the defect is healing with good quality bone occurs when the defect is can surgical procedures will not self-heal effectively. Fast serve asscaffold a good scaffold for the osteogenic cells to infiltrate. serve aswith awith formatrix the(osteoconductive osteogenic to infiltrate. filled proper (osteoconductive agent) filled with proper matrix agent) thatthat can healing quality bone occurscells when the defect is can Bone healing ismatrix further augmented in cells the presence of Bone healing is further augmented in cells the presence of proper serve a scaffold for the osteogenic to infiltrate. serve as aas scaffold for the osteogenic to infiltrate. filled with proper (osteoconductive agent) that canproper growth factors (osteoinductive agents). Autogenous bone growth factors (osteoinductive agents). Autogenous bone is is Bone healing is further augmented in the presence of proper Bone healing is further augmented in cells the presence of proper serve as a scaffold for the osteogenic to infiltrate. considered the gold standard for treating such large osseous considered the gold standard for treating such large osseous growth factors (osteoinductive agents). Autogenous bone growth factorsis(osteoinductive agents). Autogenous is is Bone healing further augmented in the presence ofbone proper defects because it provides all the components mentioned defects because it provides all the components mentioned considered the gold standard for treating such large osseous considered the gold standard for treating such large osseous growth factors (osteoinductive agents). Autogenous bone is above that bone regeneration. Additionally, above that are required for for bone defects because itstandard provides all the components mentioned defects because itrequired provides allfor theregeneration. components mentioned considered theare gold treating such Additionally, large osseous it poses no issues such as transmission and immuit poses nothat issues such as transmission and immuabove are for bone regeneration. Additionally, above that are required fordisease bone Additionally, defects because itrequired provides alldisease theregeneration. components mentioned nogenicity. However, the greatest limitation it poses is the nogenicity. However, the greatest limitation it poses is the it poses no issues such as disease transmission and immuit posesthat no are issues such as transmissionAdditionally, and immuabove required fordisease bone regeneration. creation of a second surgical site for harvesting the tissue creation of a second surgical site for harvesting the tissue nogenicity. However, the greatest limitation it poses is nogenicity. However, the greatest limitation it poses is the it poses no issues such as disease transmission and immu-the resulting insecond morbidity and increasing the risk for resulting inaHowever, greater morbidity and increasing risk creation of agreater second surgical site for harvesting the tissue creation of surgical site for harvesting the tissue nogenicity. the greatest limitation itthe poses isfor the infection. Availability of sufficient autogenous bone infection. Availability of sufficient autogenous may resulting in greater morbidity increasing the risk for resulting in morbidity andand increasing thebone risk formay creation of agreater second surgical site for harvesting the tissue add to in this limitation. issues have been addressed add to this limitation. These issues have been addressed infection. Availability of sufficient autogenous bone infection. Availability of These sufficient autogenous bone may resulting greater morbidity and increasing the risk formay by developing synthetic bone graft substitutes. Although by developing synthetic bone graft substitutes. Although add to this limitation. These issues have been addressed add to thisAvailability limitation. These issues autogenous have been addressed infection. of sufficient bone may synthetic bone graft substitutes provide the scaffold/matrix, synthetic graft substitutes provide the scaffold/matrix, synthetic bone graft substitutes. Although by developing synthetic bone graft substitutes. Although addby todeveloping thisbone limitation. These issues have been addressed they both the osteoinductive and osteogenic factors they lacklack both the osteoinductive and osteogenic factors bone graft substitutes provide the scaffold/matrix, synthetic bone graft substitutes provide the scaffold/matrix, by synthetic developing synthetic bone graft substitutes. Although which provide great advantage for effective bone healing. which provide great advantage for effective bone healing. they lack both the osteoinductive and osteogenic factors they lack both the osteoinductive and osteogenic factors synthetic bone graft substitutes provide the scaffold/matrix, The resorption of some of these synthetic matrices does The resorption of some of these synthetic matrices does notnot which provide great advantage for effective bone healing. which provide great advantage for effective bone healing. they lack both the osteoinductive and osteogenic factors parallel bone regeneration, thus compromising the quality parallel bone regeneration, thus compromising the quality The resorption of some of these synthetic matrices does The resorption of some of thesefor synthetic matrices does notofnotof which provide great advantage effective bone healing. tissue repair. Biological scaffolds will serve as more suitable tissue repair. Biological scaffolds will serve as more suitable parallel bone regeneration, thus compromising the quality parallel bone regeneration, thus compromising the quality The resorption of some of these synthetic matrices does notof of substitutes for autogenous bone. Orthopedic surgeons are substitutes forBiological autogenous bone. Orthopedic surgeons are tissue repair. Biological scaffolds will serve as more suitable tissue repair. scaffolds will serve as more suitable parallel bone regeneration, thus compromising the quality of now using composite grafts consisting of as osteoconductive now using composite grafts consisting of osteoconductive substitutes for autogenous bone. Orthopedic surgeons substitutes forBiological autogenous bone. Orthopedic surgeons areare tissue repair. scaffolds will serve more suitable scaffolds and some osteoinductive components to provide scaffolds and some osteoinductive components to provide now using composite grafts consisting of osteoconductive now using composite grafts consisting of osteoconductive substitutes for autogenous bone. Orthopedic surgeons are more of the required elements for bone regeneration. more of the required elements for bone regeneration. scaffolds and some osteoinductive components to provide scaffolds some osteoinductive components to provide now usingand composite grafts consisting of osteoconductive Allograft derived DBM is widely used a composite graft Allograft derived DBM is widely used as aas composite graft more ofand the required elements bone regeneration. more of the required elements for for bone regeneration. scaffolds some osteoinductive components to provide because itrequired provides the scaffold and the because itderived provides the natural scaffold and the Allograft derived DBM is widely used a composite graft Allograft DBM is natural widely used as aas composite graft more of the elements for bone regeneration. osteoinductive proteins that are found in bone. osteoinductive proteins that arescaffold found inas bone. because it provides scaffold the because itderived provides thethe natural and the Allograft DBM is natural widely used aand composite graft osteoinductive proteins that are found in the bone. osteoinductive proteins that are found in bone. because it provides the natural scaffold and Commercial DBM Preparations Commercial DBM Preparations osteoinductive proteins that are found in bone. Commercial DBM Preparations Commercial DBM Preparations Since initial studies performed Urist, Since thethe initial studies performed by by Urist, thethe Commercial DBM Preparations 21 21 Commercial DBM Preparations osteoinductivity of DBM has been well established. DBM osteoinductivity of studies DBM has been well established. Since initial performed by Urist, Since thethe initial studies performed by Urist, thethe DBM 21 21 first products products have have been been on on the the market market since since 1991. 1991. The The first DBM DBM osteoinductivity of DBM been well established. DBM DBM osteoinductivity of DBM hashas been well established. Since the initial studies performed by Urist, the 21 used used glycerol glycerol as the as the carrier carrier to improve to improve the the handling handling products have been on the market since 1991. The first DBM products have been on thehas market 1991. The firstDBM DBM osteoinductivity of DBM beensince well established. properties properties and and ensure ensure graft graft containment. containment. Today, Today, DBMs DBMs are are used glycerol as the carrier to improve the handling used glycerol the on carrier to improve handling products haveas been the market sincethe 1991. The first DBM used used in various in various surgical surgical applications applications including including those those of of properties and ensure graft containment. Today, DBMs are properties and ensure graft containment. Today, DBMs are used glycerol as the carrier to improve the handling spine, spine, maxillofacial, maxillofacial, and and trauma. trauma. Some Some used inreconstruction, various surgical applications including those of of used inreconstruction, various surgical applications including those of of properties and ensure graft containment. Today, DBMs are the the surgical surgical applications applications of DBMs of DBMs include: include: spine, reconstruction, maxillofacial, and trauma. Some spine, maxillofacial, and trauma. Some used inreconstruction, various surgical applications including those of of of the surgical applications of DBMs include: the surgical applications of DBMs include: spine, reconstruction, maxillofacial, and trauma. Some • •Filling Filling of voids of voids following following removal removal of aofbone a bone cystcyst orofor the •bone surgical applications of DBMs include: bone tumor tumor Filling of voids following removal a bone • Filling of voids following removal of aofbone cystcyst or or bone tumor bone tumor •• •Filling of voids following removal of a bone cyst or Filling Filling contained contained andand uncontained uncontained defects defects bone tumor Filling contained uncontained defects •• ••Filling contained andand uncontained defects Long Long bone bone fractures fractures • •Filling contained and uncontained defects Long bone fractures •• •Long bone fractures Tibial Tibial plateau plateau andand pilon pilon fractures fractures • •Long bone fractures Tibial plateau and pilon fractures •• •Tibial plateau and pilon fractures Talus Talus andand calcaneal calcaneal fractures fractures • •Tibial plateau and pilon fractures Talus calcaneal fractures •• •Talus andand calcaneal Condylar Condylar defects defects fractures • •Talus and calcaneal Condylar defects fractures •• •Condylar defects Distal Distal radius radius andand scaphoid scaphoid fractures fractures • •Condylar defectsand scaphoid fractures Distal radius •• •Distal radius and scaphoid fractures Supracondylar Supracondylar fractures fractures • •Distal radius and scaphoid Supracondylar fractures fractures •• •Supracondylar fractures Non-unions Non-unions • •Supracondylar Non-unions fractures •• •Non-unions Segmental Segmental defects defects with with fixation fixation • •Non-unions Segmental defects with fixation •• •Segmental defects with fixation Impaction Impaction grafting grafting • •Segmental defects with fixation Impaction grafting • Impaction grafting ©2009 ©2009 Zimmer Zimmer Inc. All Inc.rights All rights reserved reserved • Impaction grafting ©2009 Zimmer Inc.rights All rights reserved ©2009 Zimmer Inc. All reserved ©2009 Zimmer Inc. All rights reserved 2 • • Osteotomies: Osteotomies:opening openingand andclosing closingwedge, wedge,translational translational •• • Osteotomies: opening and closing wedge, translational Osteotomies: opening and closing wedge, translational Osteolytic Osteolyticdefects defects •• • Osteolytic defects Osteolytic defects Joint Jointfusions fusions •• • •• • Joint fusions Joint fusions Revisions Revisions Revisions Revisions Iliac Iliaccrest crestharvest harvestbackfill backfill •• • •• • Iliac crest backfill Iliac crestharvest harvest backfill Avascular Avascular necrosis necrosis Avascular necrosis Avascular necrosis oral Dental Dentalintraosseous, intraosseous, oraland andcranio-/ cranio-/maxillofacial maxillofacial defects • • defects Dental Dentalintraosseous, intraosseous,oral oraland andcranio-/ cranio-/maxillofacial maxillofacial defects defects • • Ridge Ridge augmentation augmentation •• • •• • Ridge augmentation Ridge augmentation Sinus Sinuslift lift Sinus lift Sinus lift Socket Socketpreservation preservation •• • Socket preservation Socket preservation With Withdental dental implant implantplacement placement • Demineralization placement • With Withdental dentalimplant implant placement Demineralization ofofbone bone powder powderresults resultsinina adry drygranular granular material material which which is is difficult difficult totohandle handle and andcontain contain within withinthe the Demineralization of bone powder results in a dry granular Demineralization of bone powder results in a dry granular graft graft site. site. To To improve improve handling handling characteristics, characteristics, material materialwhich whichisisdifficult difficulttotohandle handleand andcontain containwithin withinthe the cohesiveness cohesiveness and andprevention preventionofof dispersion dispersionfrom fromthe thegraft graft graft graftsite. site.ToToimprove improvehandling handlingcharacteristics, characteristics, site, site,the thegranular granularproduct productisismixed mixedwith withananinert inertcarrier. carrier. cohesiveness cohesivenessand andprevention preventionofofdispersion dispersionfrom fromthe thegraft graft Table Table 1 1below belowdepicts depicts some some ofofthese these carriers carriers used used inin site, the granular product is mixed with an inert carrier. site, the granular product is mixed with an inert carrier. DBMs DBMs that thatare arecurrently currentlyavailable availableinin the themarket marketalong along Table Table1 1below belowdepicts depictssome someofofthese thesecarriers carriersused usedinin with withthat that ofof Puros Puros DBM DBMPutty. Putty. in the market along DBMs that are currently available DBMs that are currently available in the market along with that ofofPuros DBM Putty. with that Puros DBM Putty. Puros DBM Putty: What isisit?it? Puros DBM Putty: What Puros DBM it? PurosDemineralized DBMPutty: Putty:What What it? (DBM) Puros Bone Matrix Demineralized Boneis Matrix (DBM)isisdonated donated Puros DBM Putty: What isis it? human allograft human allografttissue tissueintended intendedfor fortransplantation. transplantation.Each Each Puros PurosDemineralized DemineralizedBone BoneMatrix Matrix(DBM) (DBM)isisdonated donated lotlotofofPuros Putty isisprocessed from PurosDBM DBM Putty processed froma asingle singledonor donor human allograft tissue intended for transplantation. Each human allograft tissue intended for transplantation. Each and combines DBM and combines DBMprocessed processedusing usingtwo twoslightly slightly donor lot lotofofPuros PurosDBM DBMPutty Puttyisisprocessed processedfrom froma asingle single donor different methods totoprocessed provide handling different methods provideoptimal optimal handling and combines DBM using two slightly and combines DBM processed using two slightly characteristics while characteristics whileretaining retainingthe theosteoconductivity osteoconductivityand and different differentmethods methodstotoprovide provideoptimal optimalhandling handling osteoinductive potential of the active DBM. The only osteoinductive potential of the active DBM. The only characteristics characteristicswhile whileretaining retainingthe theosteoconductivity osteoconductivityand and excipient water. excipientininthe theproduct productisissterile sterile water.Puros PurosDBM DBMPutty Putty osteoinductive osteoinductivepotential potentialofofthe theactive activeDBM. DBM.The Theonly only has handling properties and bebemolded toto hasexcellent excellent handling properties andcan can moldedPutty excipient in the product is sterile water. Puros DBM excipient in the product is sterile water. Puros DBM Putty has excellent and has excellenthandling handlingproperties properties andcan canbebemolded moldedtoto DBM/Company DBM/Company Carrier Carrier ®® DBM/Company Grafton Grafton /OsteoTech, /OsteoTech,Inc., Inc., DBM/Company ® Biohorizons Biohorizons Grafton /OsteoTech, Inc., ® Grafton /OsteoTech, Inc., ®® Biohorizons DBX DBX /Synthes, /Synthes,Inc., Inc., Biohorizons ® Dentsply Dentsply DBX ® DBX/Synthes, /Synthes,Inc., Inc., ®® Dentsply Allomatrix Allomatrix Putty/Wright Putty/Wright Dentsply ® Medical MedicalTechnology, Technology, Inc. Inc. Allomatrix Allomatrix®Putty/Wright Putty/Wright ® ® ™™ Medical Technology, Inc. DynaGraft DynaGraft /DynaBlast /DynaBlast Medical Technology, Inc. ® Life ™ Putty/Integra Putty/Integra DynaGraft ® Life DynaGraft/DynaBlast /DynaBlast™ Sciences, Sciences,Keystone Keystone Putty/Integra Life Putty/Integra Life ®® Sciences, Keystone Accell Accell Connexus Connexus DBM/ DBM/ Sciences, Keystone ® Integra Integra Life Life Sciences Sciences Accell AccellConnexus Connexus®DBM/ DBM/ ® Life ® Integra Sciences Accell Accell DBM DBM 100/Integra 100/Integra Integra Life Sciences ® Life LifeSciences Sciences Accell Accell®DBM DBM100/Integra 100/Integra Life Sciences Puros Puros DBM DBM Putty Putty/ RTI / RTI Life Sciences Biologics, Biologics, Zimmer Zimmer Puros DBM Putty / RTI Puros DBM Putty / RTI Biologics, Biologics,Zimmer Zimmer fitfitvarious variousdefect defectshapes shapesand andsizes. sizes.ItItalso alsoprovides providesgood good graft containment containment properties properties resisting resisting irrigation. irrigation. AsAs it itisis fitgraft various defect shapes and sizes. It also provides good fit various defect shapes and sizes. It also provides good supplied supplied ininopen openbore boredispensers dispensersand andjars jarsand andstored storedatat graft graftcontainment containmentproperties propertiesresisting resistingirrigation. irrigation.AsAsit itisis controlled controlled room room temperature temperature (15-25°C), (15-25°C), the the product product isis supplied suppliedininopen openbore boredispensers dispensersand andjars jarsand andstored storedatat ready readyfor forimmediate immediate use usewithout without the theneed need for for any any is controlled room temperature (15-25°C), the product controlled room temperature (15-25°C), the product is preparation. preparation. Like Likeallallother otherDBMs, DBMs,it need itisisrestricted restricted toto ready readyfor forimmediate immediateuse usewithout withoutthe the needfor forany any homologous homologoususe usefor forrepair, repair,DBMs, replacement, replacement, ororreconstruction reconstruction preparation. Like all other it is restricted preparation. Like all other DBMs, it is restrictedtoto ofofmusculoskeletal musculoskeletal defects defectswithout withoutananintrinsic intrinsicneed needfor for homologous homologoususe usefor forrepair, repair,replacement, replacement,ororreconstruction reconstruction load bearing, bearing,bybyorordefects ononthe theorder orderofofaan alicensed licensed practitioner. practitioner. ofload musculoskeletal without intrinsic need for of musculoskeletal defects without an intrinsic need for load bearing, byby orto on ofofabreakdown load bearing, or onthe theorder order alicensed licensedpractitioner. practitioner. Water Water isisknown known to facilitate facilitate protein protein breakdown because because it itaids aidsininkeeping keepingproteases proteasesin ina ahydrated hydratedstate. state.This Thismay may Water Waterisisknown knowntotofacilitate facilitateprotein proteinbreakdown breakdownbecause because result in inprogressive progressive loss lossofof osteoinductive osteoinductive potential potential it result aids in keeping proteases in a hydrated state. This may it aids in keeping proteases in a hydrated state. This22may of ofDBMs DBMsthat thatare arereconstituted reconstituted with withwater. water.Han Hanet.al. et.al.22 result resultininprogressive progressiveloss lossofofosteoinductive osteoinductivepotential potential22 demonstrated that that hydrated hydratedDBM DBMloses losesitsitsosteoinductive osteoinductive ofdemonstrated ofDBMs DBMsthat thatare arereconstituted reconstitutedwith withwater. water.Han Hanet.al. et.al.22 potential potentialwhen when stored stored atatororabove aboveroom roomtemperature temperature for forasas demonstrated that hydrated DBM loses its osteoinductive demonstrated that hydrated DBM loses its osteoinductive little littleasas5 when 5weeks. weeks. ToToaddress address this thisissue, issue,Puros PurosDBM DBMPutty Putty potential stored at or above room temperature for potential when stored at or above room temperature forasas has hasbeen beentested testedinTo inthe theininvivo vivoratratectopic ectopic assay assayand andshown shown little littleasas5 5weeks. weeks. Toaddress addressthis thisissue, issue,Puros PurosDBM DBMPutty Putty totomaintain maintain osteoinductive osteoinductive potential potential over over the theperiod period ofofitsits has been tested in the in vivo rat ectopic assay and shown has been tested in the in vivo rat ectopic assay and shown shelf life lifewhen whenstored storedasasindicated. indicated. over the period of its toshelf tomaintain maintainosteoinductive osteoinductivepotential potential over the period of its shelf life when asasindicated. shelf life when stored indicated. Patient Patient safety safety isstored isof ofparamount paramount importance importanceduring duringallall phases phasesof ofDBM DBMprocessing processingand andhandling. handling.Each Eachlotlot ofof Patient Patientsafety safetyisisofofparamount paramountimportance importanceduring duringallall DBM DBMisof isobtained obtained from froma asingle single human humandonor donor and and isof isnot not phases DBM processing and handling. Each lot phases of DBM processing and handling. Each lot of mixed mixed with with other other donors donors in in any any formulation. formulation. The The tissue tissue DBM DBMisisobtained obtainedfrom froma asingle singlehuman humandonor donorand andisisnot not donors donors are arerigorously rigorously screened screenedin incompliance compliancewith withAATB AATB mixed mixedwith withother otherdonors donorsininany anyformulation. formulation.The Thetissue tissue standards standards and andFDA FDAregulations. regulations. InInaddition additionPuros PurosDBM DBM donors donorsare arerigorously rigorouslyscreened screenedinincompliance compliancewith withAATB AATB Putty Puttyisisterminally terminally sterilized sterilizedvia via gamma gamma irradiation irradiation asasthe the standards and FDA regulations. In addition Puros DBM standards and FDA regulations. In addition Puros DBM final finalstep step ininthe theproprietary proprietary Cancelle Cancelle SPSPirradiation DBM DBMProcess. Process. Putty is terminally sterilized via gamma as the Putty is terminally sterilized via gamma irradiation as the final step proprietary final stepininthe the proprietaryCancelle CancelleSPSPDBM DBMProcess. Process. Proprietary Processing Proprietary Processing Proprietary Processing ProprietaryProcessing Processing Carrier Glycerol Glycerol Carrier Glycerol Glycerol Sodium SodiumHyaluronate Hyaluronate Sodium SodiumHyaluronate Hyaluronate Calcium CalciumSulfate Sulfate Calcium Sulfate Calcium Sulfate Reverse ReversePhase PhaseMedium Medium Reverse Phase Medium Reverse Phase Medium Reverse ReversePhase PhaseMedium Medium Reverse ReversePhase PhaseMedium Medium Soluble SolubleDBM DBM Soluble SolubleDBM DBM Human HumanDBM DBM Human HumanDBM DBM Figure Figure 1. 1. Puros Puros DBM DBM Process Process flowchart flowchart Figure 1.1. Puros Process flowchart Figure PurosDBM DBM Process flowchart Figure 1. Puros DBM Process flowchart * DBM * DBM induced induced bone bone formation formation when when implanted implanted in in anan athymic athymic ratrat assay. assay. Findings Findings from from anan animal animal model model areare notnot necessarily necessarily predictive predictive of of human human clinical clinical results. results. * DBM induced bone formation when implanted in an athymic rat assay. Findings * DBM induced bone formation when implanted in an athymic rat assay. Findings from an animal model are not necessarily predictive of human clinical results. from an animal model are not necessarily predictive of human clinical results. Table Table 1. 1. Commercially Commercially available available DBM DBM Putties* Putties* and and the the carrier carrier utilized Table 1.utilized Commercially availableDBM DBMPutties* Putties* Table 1. Commercially available and the Table 1. Commercially available DBM Putties* and the * All * All trademarks trademarks are are thethe property property of of their their respective respective owners owners and and allall competitive competitive and the carrier utilized carrier utilized carrier utilized information information cancan bebe found found onon their their respective respective web web sites sites * All trademarks are the property of their respective owners and all competitive * All trademarks are the property of their respective owners and all competitive information can be found on their respective web sites information can be found on their respective web sites ©2009 ©2009 Zimmer Zimmer Inc.Inc. AllAll rights rights reserved reserved ©2009 Zimmer Inc. All rights reserved ©2009 Zimmer Inc. All rights reserved 3 Step 1: 1: Strict Donor Screening andand Testing Step Strict Donor Screening Testing Step 1: Strict Donor Screening and Testing Step 1:processing Strict Donor Screening and Testing is Before anyany tissue, a risk assessment Step 1:processing Strict Donor Screening and Testing is Before tissue, a risk assessment performed on every potential donor, family members areare Before processing any tissue, a risk assessment is performed on every potential donor, family members Before processing anymedical tissue, arecords risk assessment is interviewed, the donor’s are evaluated, performed on every potentialmedical donor, records family members are interviewed, donor’s aremembers evaluated, onthe every potential donor, isfamily are andperformed if necessary, thethe donor’s physician consulted. Blood interviewed, the donor’s medical records are evaluated, and if necessary, donor’s physician is consulted. Blood interviewed, the donor’s medical records are evaluated, samples from donors areare tested for for thethe presence of ofBlood and if necessary, the donor’s physician is consulted. samples from donors tested presence and if necessary, the donor’s physician is consulted. Blood infectious diseases, and Hepatitis B &B C. samples from donorsincluding are testedHIV for theand presence of infectious diseases, including HIV Hepatitis & C. samples from donors are tested for the presence of infectious diseases, including HIV and Hepatitis B & C. RTIinfectious uses many different review processes andand tests, diseases, including HIV and Hepatitis B & C. RTI uses many different review processes tests, including but not limited to: RTIincluding uses many different review processes and tests, but not limited to: RTI uses many different review processes and tests, including but/ Social not limited to: Evaluation • including but/ Social not History limited to: Evaluation •Medical Medical History • oMedical / Social History Evaluation Family/Next-of-Kin interview • oMedical / Social History Evaluation Family/Next-of-Kin interview oo Family/Next-of-Kin interview record review ooMedical/Hospital Family/Next-of-Kin interview Medical/Hospital record review oo Medical/Hospital record review Behavioral/Lifestyle risk assessment Medical/Hospital record oo Behavioral/Lifestyle riskreview assessment oo Behavioral/Lifestyle risk assessment Examiner/Coroner’s report (autopsy ooMedical Behavioral/Lifestyle risk assessment Medical Examiner/Coroner’s report (autopsy when available) o report, Medical Examiner/Coroner’s report (autopsy report, when available) o Medical Examiner/Coroner’s report (autopsy whenpathology available)and radiology reports o oreport, Laboratory, report, whenpathology available)and radiology reports Laboratory, o Laboratory, pathology and radiology reports • •Serological Testing oSerological Laboratory, pathology and radiology reports Testing • oSerological Testing HCV Antibody • oSerological Testing HCV Antibody oo HCV Antibody Surface Antigen ooHBV HCV Antibody HBV Surface Antigen oo HBV Surface Antigen HIV 1 & 2 Antibody HBV1Surface Antigen oo HIV & 2 Antibody oo HIV 1 & 2 Antibody Total Antibody ooHBV HIV 1Total &Core 2 Antibody HBV Core Antibody oo HBV Total Core Antibody I &II Antibody ooHTLV HBV Total Core Antibody HTLV I &II Antibody oo HTLV I &II Antibody for for Syphilis ooRPR HTLV I &II Antibody RPR Syphilis oo RPR for Syphilis ooHIV-1/NAT RPR for Syphilis HIV-1/NAT oo HIV-1/NAT ooHCV/NAT HIV-1/NAT HCV/NAT o HCV/NAT • •Microbiological oMicrobiological HCV/NATTesting Testing • oMicrobiological Testing Pre-processing culturing: Performed before • oMicrobiological Testing Pre-processing culturing: Performed before processing begins; removes potentially unsuitable o Pre-processing culturing: Performed before processing begins; removes potentially unsuitable otissue Pre-processing culturing: Performed before from process processing begins; removes potentially unsuitable tissue from process processing begins; removes potentially unsuitable from process o otissue Environmental controls: Monitors cleanliness of of tissue from process Environmental controls: Monitors cleanliness processing environment o Environmental controls: Monitors cleanliness of processing environment o Environmental controls: Monitors cleanliness of processing environment TheThe final determination of donor eligibility is made by by processing environment final determination of donor eligibility is made RTI’s medical director-a physician-utilizing all all TheRTI’s final medical determination oflicensed donor eligibility is made by director-a licensed physician-utilizing The final determination of donor eligibility is made by available, relevant information. RTI’s medicalrelevant director-a licensed physician-utilizing all available, information. RTI’s medical director-a licensed physician-utilizing all available, relevantand information. Step 2: Milling Demineralization available, relevantand information. Step 2: Milling Demineralization Step 2: Milling and Demineralization a. Step Milling Step 2: Milling and 2: Milling andDemineralization Demineralization a. Milling a. Milling Bone from donors thatthat pass thethe above criteria is processed a. Milling Bone from donors pass above criteria is processed to make Puros DBM Putty. Tissue processing forprocessed each Bone from donors that pass the above criteria is to make Puros DBM Putty. Tissue processing for each Bone from donors that pass the above criteria is processed donor lotPuros occurs in controlled, clean-room environments to make DBM Putty. Tissue processing for each donor lot occurs in controlled, clean-room environments to make Puros DBM Putty. Tissue processing for and thelot room is cleaned between donors. The firstfirsteach donor occurs in cleaned controlled, clean-room environments and the room is between donors. The donor lot occurs in controlled, clean-room environments processing step involves milling the cleaned and the room isstep cleaned between donors. Thebone firstto to processing milling the cleaned and the room is involves cleaned between donors. Thebone first optimal particle size so that it retains both processing step involves milling the cleaned bone to optimal particle size so that it retains both bone processing step involves milling the cleaned to osteoconductive and osteoinductive properties. optimal particle size so that it retains both osteoconductive and so osteoinductive optimal particle size that it retainsproperties. both osteoconductive and osteoinductive properties. osteoconductive and osteoinductive properties. Donor bone tissue is milled such thatthat Puros DBM Putty hashas Donor bone tissue is milled such Puros DBM Putty DBM particles that fall within size ranges shown to produce Donor bone tissue that is milledwithin such that Puros DBM Putty has DBM particles size ranges shown toPutty produce Donor bone tissue isfall milled such that Puros has bone growth (125-1000 µm), thus providing anDBM DBM particles that fall within size ranges shown to produce bone growth (125-1000 µm), thus providing an DBM particles that fall 24 within size ranges shown to produce osteoconductive matrix. 24 thus providing an bone growth (125-1000 µm), osteoconductive matrix. bone growth (125-1000 µm), thus providing an 24 osteoconductive matrix. 24 b. osteoconductive Demineralization matrix. b. Demineralization b. Demineralization Bone is aisnatural reservoir of growth factors thatthat areare b. Demineralization Bone a natural reservoir of growth factors required for its formation. In native bone, these factors Bone is a natural reservoir of growth factors that are areare required its formation. In native bone, these Bone is afor natural reservoir of growth factors thatfactors are sequestered within the matrix and remain inaccessible required for its formation. In native bone, these factors are sequestered within the matrix and remain inaccessible required for its formation. In native bone, these factors are unless the matrix is broken down. Demineralization is the sequestered within the matrix and remain inaccessible unless the matrix isthe broken down. Demineralization is the sequestered within matrix and remain inaccessible first step during breakdown, which improves unless the matrixmatrix ismatrix broken down. Demineralization is the first step breakdown, which improves unless theduring matrix is broken down. Demineralization is the accessibility of the growth factors. While several methods firstaccessibility step during of matrix breakdown, which improves the growth factors. While several methods first step during matrix breakdown, which improves cancan be employed demineralizing bone, thethe most accessibility of theforgrowth factors. While several methods be employed demineralizing bone, most accessibility of theforgrowth factors. While several common and effective way utilizes mineral acids as methods can be employed for demineralizing bone, the most common and effective way utilizes mineral acids as can be employed for demineralizing bone, the most 7 Proper cancan result in described by by Urist. 7 way demineralization common and effective utilizes mineral acids as result Proper demineralization described Urist. common and effective way utilizes mineral acids as in 7 which retains all or most of its high quality Proper demineralization canofresult in described bymatrix Urist. 7 which high quality matrix retains all or most its demineralization can result in described bypotential. Urist. Proper osteoinductive AATB high quality matrixpotential. whichAccording retains all to or the most ofAATB itsstandards, osteoinductive According to the standards, highshould qualityhave matrix which retains allofor8% most of itsA study DBMs a calcium content or less. osteoinductive potential. According to the AATB DBMs should have a calcium content ofthe 8% or standards, less. A study osteoinductive potential. According to AATB standards, further examining calcium levels within thisthis DBMs should haveresidual a residual calcium content of 8% or less. Arange study further examining calcium levels within range DBMs should have a calcium content of 8% or less. A demonstrated thatthat the optimal residual calcium is study further examining residual calcium levels withincontent this range demonstrated the optimal residual calcium content is further examining residual calcium levels within this range less than 1%.1%. Puros Putty is therefore processed so demonstrated thatPuros theDBM optimal residual calcium content is as less than DBM Putty is therefore processed so as demonstrated that the optimal 25residual calcium content is to within this optimal range. 25 lessfall than 1%. Puros DBM Putty is therefore processed so as to within optimal lessfall than 1%. this Puros DBMrange. Putty is therefore processed so as 25 to fall within this optimal range. 25 TheThe Cancelle SP DBM Process is validated to thethe to fall within this optimal range.is validated inactivate Cancelle SP DBM Process to inactivate following model, relevant and challenge viruses providing Thefollowing Cancellemodel, SP DBM Process validated viruses to inactivate the relevant andis challenge providing The Cancelle SPofDBM Process is validated to inactivate the an additional level safety to challenge the recipient of the Puros following model, relevant and viruses providing an additional level of safety to the recipient of the Puros following model, relevant and challenge viruses providing DBM Putty implant: an additional level of safety to the recipient of the Puros DBM Putty implant: an additional level of safety to the recipient of the Puros DBM Putty implant: • DBM Viral Diarrhea Virus (BVDV) Model Putty implant: •Bovine Bovine Viral Diarrhea Virus (BVDV) Model • Bovine Viral Diarrhea Virus (BVDV) Model Human Immunodeficiency Virus (HIV) •o Bovine Viral Diarrhea Virus (BVDV) Model o Human Immunodeficiency Virus (HIV) o Human Immunodeficiency Virus (HIV) o o Virus Model (HCV) oHepatitis HumanCImmunodeficiency Virus (HIV) Hepatitis C Virus Model (HCV) o Hepatitis C Virus Model (HCV) o o T-lymphotropic Virus (HTLV) Hepatitis C Virus Model (HCV) oHuman Human T-lymphotropic Virus (HTLV) o Human T-lymphotropic Virus (HTLV) • •Pseudorabies Virus (PrV) Model o Human T-lymphotropic Virus Pseudorabies Virus (PrV) Model (HTLV) • Pseudorabies (PrV) Model BVirus Virus (HBV) •o Pseudorabies (PrV) Model oHepatitis Hepatitis BVirus Virus (HBV) o Hepatitis B Virus (HBV) • •Human Poliovirus (Polio-1) Challenge o Hepatitis B Virus (HBV) Human Poliovirus (Polio-1) Challenge •• Human Poliovirus (Polio-1) Challenge Porcine Parvovirus (PPV) Challenge •• Human Challenge Porcine Poliovirus Parvovirus(Polio-1) (PPV) Challenge •Step Porcine Parvovirus (PPV) Challenge 3:Porcine Test for Osteoinductive Potential •Step Parvovirus (PPV) Challenge 3: Test for Osteoinductive Potential Step 3: Test for Osteoinductive Potential TheStep osteoinductive potential of DBM cancan be highly variable 3: Test for Osteoinductive Potential The osteoinductive potential of DBM be highly variable Step 3: Test for Osteoinductive Potential depending on the individual donor, processing methods used The osteoinductive potential of DBM can be highly variable depending on the individual donor, processing methods used The osteoinductive potential of DBM can be highly variable 26 to demineralize andand thedonor, carrier used to make DBM. depending on thebone individual processing methods used26 to demineralize bone the carrier used to make DBM. depending on the individual donor, processing methods used 26 Because variability donors exists, each lot lot of DBM to demineralize boneamong and the carrier used to make DBM. 26 Because variability among donors exists, each of DBM to demineralize bone and the carrier used to make DBM. must be assessed to among assure osteoinductive potential. Because variability donors exists, each lot of DBM must be assessed to assure osteoinductive potential. Because variability among donors exists, each lot of DBM must be bone assessed to assure osteoinductive potential. Ectopic formation in an rodent muscle pouch must be bone assessed to assure osteoinductive potential. Ectopic formation in athymic an athymic rodent muscle pouch is aisstandard method of demonstrating the osteoinductive Ectopic bone formation in an athymic rodent muscle pouch a standard method of demonstrating the osteoinductive Ectopicof bone formation in an athymic rodent muscle pouch properties DBM. Although a few in vitro assays have is aproperties standard method ofAlthough demonstrating the osteoinductive of method DBM. a few in vitro assays have is a standard of demonstrating the osteoinductive been validated against these animal in vivo properties of DBM. Although a animal few models, in vitro assays have been validated against these models, in vivo properties of DBM. Although a few instandard vitro assays haveit testing in rodents is considered the gold because been validated against these animal models, in vivo testing in rodents is considered the gold standard because it been validated against these animal models, in vivo evaluates thethe osteoinductive potential ofstandard the graft material testing in rodents is considered the gold because it evaluates osteoinductive potential of the graft material testing inentire rodents is considered the gold standard because it through the cascade ofpotential steps leading the formation evaluates the osteoinductive of thetograft material through the entire cascade ofpotential steps leading tograft the formation evaluates the osteoinductive of the of bone. In the presence of of an osteoinductive in material ainnonthrough the entire cascade leading to graft thegraft formation of bone. In the presence ofsteps ansteps osteoinductive a nonthrough the entire cascade of leading to the formation bony site, a process similar to endochondral bone formation of bone. In the presence of an osteoinductive graft in a nonbony site,Inathe process similar to osteoinductive endochondral bone formation of bone. presence of an graft in a nonis observed stem cells differentiate bony site, a where processmesenchymal similar to endochondral bone formation is observed stem cells differentiate bony site, a where processmesenchymal similar to endochondral bone formation is observed where mesenchymal stem cells differentiate is observed where mesenchymal stem cells differentiate ©2009 Zimmer Inc. All rights reserved ©2009 Zimmer Inc. All rights reserved ©2009 Zimmer Inc. All rights reserved ©2009 Zimmer Inc. All rights reserved 4 into into cartilage cartilage cells cells (chondrocytes) (chondrocytes) and and bone-forming bone-forming cells cells (osteoblasts), (osteoblasts), producing producing new new bone. bone.and Bone Bone grafts grafts that that do do not not intocartilage cartilagecells cells (chondrocytes) and bone-forming cells into (chondrocytes) bone-forming cells trigger trigger this this process process of of endochondral endochondral ossification ossification may may be be (osteoblasts), producing new bone. Bone grafts that do not (osteoblasts), producing new bone. Bone grafts that do not into cartilage cells (chondrocytes) and bone-forming cells biocompatible biocompatible but but are are not not osteoinductive. osteoinductive. triggerthis thisprocess process ofendochondral endochondral ossification may be trigger of ossification may (osteoblasts), producing new bone. Bone grafts that dobe not biocompatible butare are notosteoinductive. osteoinductive. biocompatible but not trigger this process of endochondral ossification may be Each Each lot lot of of DBM DBM used used in in Puros Puros DBM DBM Putty Putty isis tested tested biocompatible but are not osteoinductive. for for osteoinductive osteoinductive potential potential after afterDBM irradiation. irradiation. Each lotof ofDBM DBMused used inPuros Puros DBM PuttyisBiological isBiological tested Each lot in Putty tested activity activity assessed (qualitatively (qualitatively and and quantitatively) quantitatively) using forosteoinductive osteoinductive potential afterDBM irradiation. Biological for potential after irradiation. Each lotisis ofassessed DBM used in Puros Putty isBiological tested using 77 the the Urist Urist athymic athymic rat rat model. model. This This in in vivo vivo model model has has been been activity is assessed (qualitatively and quantitatively) using activity is assessed (qualitatively and quantitatively) using for osteoinductive potential after irradiation. Biological 77 utilized utilized by by RTI RTI Biologics Biologics to to identify identify DBM with with has theUrist Uristis athymic rat model. Thisand inDBM vivomodel model hasbeen been the athymic rat model. This in vivo activity assessed (qualitatively quantitatively) using 7byosteoinductivity acceptable acceptable osteoinductivity for use useinin inDBM all all Puros Puros DBM utilized RTIBiologics Biologics tofor identify DBM withDBM utilized RTI to identify with the Uristby athymic rat model. This vivo model has been Putty Putty products. products. The The implants implants are are extracted extracted after afterDBM 28 28 days days acceptable osteoinductivity for use inDBM allPuros Puros DBM acceptable use in all utilized by osteoinductivity RTI Biologics tofor identify with and and analyzed analyzed histologically histologically for osteoinductivity osteoinductivity and and Putty products. Theimplants implants are extracted afterDBM 28days days Putty products. The are extracted after 28 acceptable osteoinductivity for use in all Puros inflammatory inflammatory responses responses in in accordance accordance with withafter the the scoring scoring andanalyzed analyzed histologically for osteoinductivity and and histologically for and Putty products. The implants areosteoinductivity extracted 28 days 27 27 system system of of Edwards Edwards et et al. al.in (1998). (1998). In In addition, addition, bone inflammatory responses in accordance withthe thebone scoring inflammatory responses accordance with scoring and analyzed histologically for osteoinductivity and 27 27 maturity maturity also also scored scored in inin(1998). accordance accordance with with Katz et et al. al. Inaddition, addition, bone systemof ofisisEdwards Edwards al. (1998). system etetal. In inflammatory responses accordance withKatz thebone scoring 28 28 27 that Only Only those lots of DBM DBM thataddition, pass passKatz this thisbone in in vivo (2006). (2006). maturity is alsothose scored inof accordance with Katz etvivo al. maturity is also scored in accordance with et al. system of Edwards etlots al. (1998). In 28 28 rat rat assay assay with with minimal inflammatory inflammatory response are further further (2006). Only those lots ofDBM DBMthat thatresponse passKatz thisare invivo vivo Only those lots of pass this in (2006). maturity is alsominimal scored in accordance with et al. 28 with processed processed into intominimal Puros Puroslots DBM DBM Putty. thatresponse ratassay assay minimal inflammatory response are further rat with inflammatory further (2006). Only those of Putty. DBM pass thisare in vivo processed intominimal PurosDBM DBM Putty. response are further processed into Puros Putty. rat assay with inflammatory Delivering patient patient safety safety Delivering processed into Puros DBM Putty. Deliveringpatient patientsafety safety Delivering Delivering safety The primary primarypatient goal isis to to ensure ensure patient patient safety. safety.To To fulfill fulfill this this The goal Delivering patient safety goal, stringent tissue donor screening, laboratory testing goal, stringent tissue donor screening, laboratory testing Theprimary primary goal to ensure patientsafety. safety. Tofulfill fulfill this The goal isisto ensure patient To this and tissue preparation processes validated toTo address and tissue preparation processes validated to address goal, stringent tissue donor screening, laboratory testing goal, stringent tissue screening, laboratory testing The primary goal is todonor ensure patient safety. fulfill this the potential for disease transmission are employed for the for disease transmission are employed for andpotential tissue preparation processes validated toaddress address and tissue preparation processes validated to goal, stringent tissue donor screening, laboratory testing Puros DBM Putty. As discussed discussed earlier, the redundant Puros DBM Putty. As earlier, redundant thepotential potential fordisease disease transmission arethe employed for the for transmission are employed for and tissue preparation processes validated to address safeguards include a) screening screening donor medical history by safeguards include a) donor medical history Puros DBM Putty. As discussed earlier, the redundant Puros DBM Putty. As discussed earlier, the redundant the potential for disease transmission are employed forby evaluating medical records and interviewing interviewing family and evaluating records and family and safeguards include a) screening donormedical medical history by safeguards include a) screening donor history by Puros DBMmedical Putty. As discussed earlier, the redundant next-of-kin, b) conducting an extensive panel of next-of-kin, b) conducting an extensive panel of evaluating medical records and interviewing family and evaluating medical records and interviewing family and safeguards include a) screening donor medical history by serological and microbiological tests, c) c)panel demineralizing serological and microbiological tests, demineralizing next-of-kin, b)conducting conducting anextensive extensive panel of and next-of-kin, b) of evaluating medical recordsan and interviewing family using validated process tested using model viruses in using validated process tested using model viruses serological and microbiological tests, c)panel demineralizing serological and microbiological tests, c) demineralizing next-of-kin, b) conducting an extensive of in 23 23 accordance withmicrobiological FDAguidance guidance accordance with FDA ,, and and d) d)demineralizing terminal terminal usingvalidated validated process testedusing using model virusesin in using process tested model viruses serological and tests, c) 23 23 sterilization sterilization of of product product using using low-temperature, low-dose low-dose accordance with FDAguidance guidance and d)terminal terminal accordance with FDA ,,and d) using validated process testedlow-temperature, using model viruses in gamma gamma irradiation irradiation to to achieve achieve alow-temperature, a23 validated validated 10 10-6-6 sterility sterility sterilization ofproduct product usinglow-temperature, low-dose sterilization of using low-dose accordance with FDA guidance , and d) terminal assurance assurance level. level. These These redundant redundant safeguards safeguards provide aa gammairradiation irradiation toachieve achieve validated 10-6-6provide sterility gamma to aavalidated 10 sterility sterilization of product using low-temperature, low-dose -6 provide high high level level of of confidence confidence that that patients patients will will10 receive receive safe, safe, assurance level. These redundant safeguards assurance level. These redundant safeguards provide aa gamma irradiation to achieve a validated sterility high level quality quality tissue. levellevel. oftissue. confidence thatpatients patients willreceive receive safe, high of confidence that will safe, assurance These redundant safeguards provide a highlevel quality tissue. quality high oftissue. confidence that patients will receive safe, General General Information Information high quality tissue. GeneralInformation Information General For For further further information information regarding regarding Puros Puros DBM DBM products, products, General Information please please refer refer to to the the Instructions Instructions For ForPuros Use. Use. DBM Forfurther further information regarding Puros DBMproducts, products, General Information For information regarding please referinformation tothe theInstructions Instructions ForPuros Use. DBM products, please refer to For Use. For further regarding RTI RTI Biologics Biologics Inc. Inc. Credentials Credentials please refer to the Instructions For Use. RTIBiologics BiologicsInc. Inc.Credentials Credentials RTI •• RTI RTI Biologics Biologics isis accredited accredited by by the theAmerican American RTI Biologics Inc. Credentials Association of ofis Tissue Tissue Banks Banksby (AATB) (AATB) for for processing, processing, RTIBiologics Biologics is accredited by theAmerican American •• Association RTI accredited the storage and and distribution distribution of of skin, skin, pericardium pericardium and and Association ofis Tissue Banks (AATB) forprocessing, processing, Association of Tissue Banks (AATB) for • storage RTI Biologics accredited by the American musculoskeletal musculoskeletal tissue tissueBanks for for transplantation and andand research research storageand anddistribution distribution oftransplantation skin, pericardium and storage of skin, pericardium Association of Tissue (AATB) for processing, musculoskeletal tissuefor for transplantation and research musculoskeletal tissue and research storage and distribution of transplantation skin, pericardium and •• RTI RTI Biologics Biologics isis registered registered as as aaTissue Tissue Establishment Establishment musculoskeletal tissue for transplantation and research with the the U.S. U.S. Food Food and and Drug Drug Administration (FDA). (FDA). RTIBiologics Biologics registered asAdministration TissueEstablishment Establishment •• with RTI isisregistered as aaTissue withBiologics theU.S. U.S.Food Food andDrug Drug Administration (FDA). the and (FDA). • with RTI is registered asAdministration a Tissue Establishment •• RTI RTI Biologics Biologics isis registered registered as as aa Medical Medical Device Device with the U.S. Food and Drug Administration (FDA). Manufacturer with the the FDA. FDA. RTIBiologics Biologicswith isregistered registered asaaMedical MedicalDevice Device •• Manufacturer RTI is as Manufacturer with theFDA. FDA. the • Manufacturer RTI Biologics with is registered as a Medical Device •• RTI RTI Biologics’ Biologics’quality quality system system isis certified certified to to ISO ISO Manufacturer with the FDA. 13485:2003 (with (with CMDCAS CMDCAS for Canada) Canada)to Medical Medical RTIBiologics’ Biologics’ quality systemfor certified to ISO •• 13485:2003 RTI quality system isiscertified ISO Devices Quality Quality Management Management Systems. 13485:2003 (with CMDCAS for Canada)to Medical 13485:2003 (with CMDCAS Canada) Medical • Devices RTI Biologics’ quality systemfor isSystems. certified ISO DevicesQuality Quality Management Systems. Devices Systems. 13485:2003 (withManagement CMDCAS for Canada) Medical Devices Quality Management Systems. •• •• • RTI Biologics Biologics isis registered registered with with Health Health Canada Canada for for RTI Human Cells,Tissue Tissue and Organs Organs forTransplantation Transplantation Human Cells, and for RTIBiologics Biologics isregistered registered withHealth Health Canadafor for RTI is with Canada (CTO). (CTO). Human Cells,Tissue Tissue andOrgans Organs forTransplantation Transplantation Human Cells, and for RTI Biologics is registered with Health Canada for (CTO). Cells, Tissue and Organs for Transplantation (CTO). Human RTI Biologics Biologics isis licensed licensed as as aaTissue Tissue Bank Bank by by the the •• RTI (CTO). following states: states: RTIBiologics Biologics licensedas asaaTissue TissueBank Bankby bythe the •• following RTI isislicensed following states: states: • following RTI Biologics is licensed as a Tissue Bank by the Florida oo Florida following states: Florida oo Florida Maryland oo Maryland o Florida Maryland oo Maryland California oo California o Maryland California oo California NewYork York oo New o California NewYork York oo New Other state state registrations registrations as as applicable: applicable: oo Other o New York (Delaware, Illinois, and and Oregon) Oregon) Illinois, Otherstate stateregistrations registrations asapplicable: applicable: oo (Delaware, Other as (Delaware, Illinois,and andOregon) Oregon) Illinois, o (Delaware, Other state registrations as applicable: RTI Biologics Biologics isis licensed licensed as as aa Medical Medical Device Device •• RTI (Delaware, Illinois, and Oregon) Manufacturer with the State State of Florida.Device the Florida. RTIBiologics Biologicswith islicensed licensed asaof aMedical Medical Device •• Manufacturer RTI is as Manufacturer with theState State ofMedical Florida.Device the Florida. • Manufacturer RTI Biologics with is licensed as aof RTI Biomedical Biomedical Laboratory Laboratory holds: holds: •• RTI Manufacturer with the State of Florida. RTIBiomedical BiomedicalLaboratory Laboratoryholds: holds: •• RTI FDARegistration Registration oo FDA • RTI Biomedical Laboratory holds: FDARegistration Registration oo FDA Clinical Laboratory Laboratory Improvement ImprovementAmendments Amendments oo Clinical o FDA Registration (CLIA) Certificate of ComplianceAmendments (Federal) Compliance (Federal) ClinicalCertificate Laboratoryof Improvement Amendments oo (CLIA) Clinical Laboratory Improvement (CLIA)Certificate Certificateof of ComplianceAmendments (Federal) Compliance (Federal) o (CLIA) Clinical Laboratory Improvement State of of Florida Florida Clinical Clinical Laboratory Laboratory License License oo State (CLIA) Certificate of Compliance (Federal) Stateof ofFlorida FloridaClinical ClinicalLaboratory LaboratoryLicense License oo State NewYork York State State Department Department of of Health Health Clinical Clinical oo New o State of Florida Clinical Laboratory License Laboratory Permit Permit NewYork York State State Departmentof ofHealth HealthClinical Clinical oo Laboratory New Department Laboratory Permit Permit o Laboratory New York State Department of Health Clinical All lab lab tests tests are are performed performed using using kits kits approved approved by by the the All Laboratory Permit FDA for donor screening and cadaveric specimens, FDA for donor and cadaveric specimens, ifif Alllab lab tests arescreening performed using kitsapproved approved bythe the All tests are performed using kits by applicable. Laboratory procedures comply withby thethe applicable. Laboratory procedures comply with the kit FDA for donor screening and cadaveric specimens, if FDA for donor and cadaveric specimens, ifkit All lab tests arescreening performed using kits approved manufacturer’s instructions for usecomply regarding testing manufacturer’s instructions for use regarding testing applicable. Laboratory procedures comply with thekit applicable. Laboratory procedures with the FDA for donor screening and cadaveric specimens, ifkit protocol, specimen typeprocedures andfor specimen handling/storage protocol, specimen type and specimen handling/storage manufacturer’s instructions for usecomply regarding testing manufacturer’s instructions use regarding testing applicable. Laboratory with the kit requirements. requirements. protocol,specimen specimen typeand andfor specimen handling/storage protocol, type specimen handling/storage manufacturer’s instructions use regarding testing requirements. requirements. protocol, specimen type and specimen handling/storage Zimmer, Inc. Inc. Credentials Credentials Zimmer, requirements. Zimmer,Inc. Inc.Credentials Zimmer, Zimmer, tissue Inc.Credentials Credentials Zimmer’s tissue bank establishment, establishment, Zimmer Zimmer Spine, Spine, isis Zimmer’s bank Zimmer, Inc. Credentials registered with the U.S. Food and and Drug Drug Administration, registered with U.S. Food Administration, Zimmer’stissue tissuethe bank establishment, Zimmer Spine,isis Zimmer’s bank establishment, Zimmer Spine, and licensed as tissue bank in the the following states: and isis licensed aaU.S. tissue bank in following states: registered withas the U.S. Food and Drug Administration, registered with the Food and Drug Administration, Zimmer’s tissue bank establishment, Zimmer Spine, is and is licensed as a tissue bank in the following states: and is licensed as a tissue bank in the following states: registered with the U.S. Food and Drug Administration, •• Florida, Florida, Maryland, Maryland, California, California, New NewYork York and is licensed as a tissue bank in the following states: Florida,Maryland, Maryland,California, California,New NewYork York •• Florida, • Florida, Maryland, California, New York ©2009 ©2009Zimmer ZimmerInc. Inc.All Allrights rightsreserved reserved ©2009Zimmer ZimmerInc. Inc.All Allrights rightsreserved reserved ©2009 ©2009 Zimmer Inc. All rights reserved 5 References References References 1.1. Peterson, Peterson,B., B.,Whang, Whang,P.P.G., G.,Iglesias, Iglesias,R., R.,Wang, Wang,J.C., J.C.,and and Liberman, Liberman,J.R., J.R.,Osteoinductivity Osteoinductivityofofcommercially commerciallyavailable available deminerailized deminerailizedbone bonematrix. matrix.Preparations Preparationsininaaspine spinefusion fusion model. model.J.J.Bone BoneJoint JointSurg SurgAm. Am.86:2243-2250, 86:2243-2250,(2004) (2004) 2.2. Mulliken, Mulliken,J.,J.,Kaban, Kaban,L., L.,Glowacki, Glowacki,J.,J.,Induced Inducedosteogenesis osteogenesis- The Thebiological biologicalprinciple principleand andclinical clinicalapplications. applications.J.J.Surg. Surg.Res., Res., 37: 37: 487-496, 487-496,(1984). (1984). 3.3. Yazdi, Yazdi,M., M.,Schonfeld, Schonfeld,S., S.,Demineralized Demineralizedbone bonematrix matrixinin treatment treatmentofofperiodontal periodontaldefects. defects.AAreview reviewofofthe theliterature. literature. J.J.Western WesternSoc. Soc.Periodont., Periodont.,35: 35:105-108, 105-108,(1987). (1987). 4.4. Urist, Urist,M., M.,Strates, Strates,B., B.,Bone Boneformation formationininimplants implantsofofpartially partially and andwholly whollydemineralized demineralizedbone bonematrix. matrix.Clin. Clin.Orthop. Orthop.Rel. Rel.Res., Res., 71: 71:271-278, 271-278,(1970). (1970). 5.5. Zhang, Zhang,M., M.,Powers, Powers,R.M., R.M.,Wolfinbarger, Wolfinbarger,L., L.,Effect(s) Effect(s)ofofthe the demineralization demineralizationprocess processon onthe theosteoinductivity osteoinductivityofofdemineraldemineralized izedbone bonematrix. matrix.JJPeriodontol., Periodontol.,68: 68:1085-1092, 1085-1092,(1997). (1997). 6.6. Senn, Senn,N., N.,On Onthe thehealing healingofofaseptic asepticbone bonecavities cavitiesby by implantation implantationofofantiseptic antisepticdecalcified decalcifiedbone. bone. Amer. Amer.JJMed. Med.Sci., Sci., 98: 98:219-243, 219-243,(1889). (1889). 7.7. Urist, Urist,M., M.,Bone Boneformation formationby byautoinduction. autoinduction.Science, Science,150: 150:893893899, 899,(1965). (1965). 8.8. Urist, Urist,M., M.,Strates, Strates,B., B.,Bone Bonemorphogenetic morphogeneticprotein. protein.J.J.Dent. Dent. Res., Res.,(Suppl. (Suppl.6)6)50: 50:1392, 1392,(1971). (1971). 9.9. Urist, Urist,M., M.,DeLange, DeLange,R., R.,Finerman, Finerman,G., G.,Bone Bonecell celldifferentiation differentiation and andgrowth growthfactors. factors.Science, Science,220: 220:680 680- -686, 686,(1983). (1983). 10. 10. Mulliken, Mulliken,J.,J.,Glowacki, Glowacki,J.,J.,Kaban, Kaban,L., L.,Folkman, Folkman,J.,J.,Murray, Murray,J.,J., Use Useofofdemineralized demineralizedallogeneic allogeneicbone boneimplants implantsfor forthe thecorreccorrection tionofofmaxillofacial maxillofacialdeformities. deformities.Ann. Ann.Surg., Surg.,194: 194:366-372, 366-372, (1981). (1981). 11. 11. Tiedman, Tiedman,J.,J.,Garvin, Garvin,K., K.,Kile, Kile,T., T.,Connolly, Connolly,J.,J.,The Therole roleofofaa composite compositedemineralized demineralizedbone bonematrix matrixand andbone bonemarrow marrowinin the thetreatment treatmentofofosseous osseousdefects. defects.Orthopedics, Orthopedics,18:1153-1158, 18:1153-1158, (1995). (1995). 12. 12. Leriche, Leriche,R., R.,Policard, Policard,A., A.,eds. eds.The TheNormal Normaland andPathological Pathological Physiology PhysiologyofofBone Boneand andits itsProblems. Problems.London, London,England: England:Henry Henry Kimpton; Kimpton;(1928). (1928). 13. 13. Levander, Levander,G., G.,AAstudy studyofofbone boneregeneration. regeneration.Surg. Surg.Gynecol. Gynecol. Obstet., Obstet.,67: 67:705-714, 705-714,(1938). (1938). 21. 21. Ludwig, Ludwig,S.C., S.C.,Kowalski, Kowalski,J.M., J.M.,Boden, Boden,S.D., S.D.,Osteoinductive Osteoinductive bone bonegraft graftsubstitutes. substitutes.Eur. Eur.Spine SpineJ.,J.,9(Suppl1): 9(Suppl1):S119-S125, S119-S125, (2000). (2000). 22. 22. Han, Han,B., B.,Yang, Yang,Z., Z.,Nimni, Nimni,M., M.,Effects Effectsofofmoisture moistureand and temperature temperatureon onthe theosteoinductivity osteoinductivityofofdemineralized demineralizedbone bone matrix. matrix.J.J.Orthop. Orthop.Res., Res.,23: 23:855-861, 855-861,(2005). (2005). 23. 23. US USFood Foodand andDrug DrugAdministration Administration(FDA) (FDA)Center Centerfor forBiologics Biologics Evaluation Evaluationand andResearch Research(CBER). 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Wang, Wang,J.C., J.C.,Alanay, Alanay,A., A.,Mark, Mark,D., D.,Kanim, Kanim,L.E.A., L.E.A.,Campbell, Campbell, P.A., P.A.,Dawson, Dawson,E.G., E.G.,Liberman, Liberman,J.R., J.R., AAcomparison comparisonofof commercially commerciallyavailable availabledemineralized demineralizedbone bonematrix matrixfor forspinal spinal fusion. fusion.Eur. Eur.Spine SpineJ.,J.,16(8): 16(8):1233-1240, 1233-1240,(2007). (2007). 27. 27. Edwards, Edwards,J.T., J.T.,Diegmann, Diegmann,M.H., M.H.,Scarborough, Scarborough,N.L., N.L., Osteoinduction Osteoinductionofofhuman humandemineralized demineralizedbone: bone:characterization characterization ininaarat ratmodel. model.Clin. Clin.Orthop. Orthop.Rel. Rel.Res. Res.357: 357:219-28, 219-28,(1998). (1998). 28. 28. Katz, Katz,J.J.M., M.,Diegl, Diegl,E.E.C., C.,Nataraj, Nataraj,C., C.,Time Timecourse courseofofDBM DBM induced inducedintermuscular intermuscularendochondral endochondralossification. ossification.Orthopedic Orthopedic Research ResearchSociety SocietyMeeting Meeting(2006) (2006)abs.912. abs.912. 29. 29. Han, Han,B., B.,Yang, Yang,Z., Z.,Nimni, Nimni,M., M.,Effects Effectsofofgamma gammairradiation irradiationon on osteoconduction osteoconductionassociated associatedwith withdemineralized demineralizedbone bonematrix. matrix. J.J.Orthop. Orthop.Res., Res.,26(1): 26(1):75-82, 75-82,(2008). (2008). 30. 30. Qiu, Qiu,Q.Q., Q.Q.,Connor, Connor,J.,J.,Effect Effectofofgamma gammairradiation, irradiation,storage storage and andhydration hydrationon onosteoinductivity osteoinductivityon onDBM DBMand andDBM/AM DBM/AM composite. composite.J.J.Biomed. Biomed.Mater. Mater.Res. Res.A. A. 87(2): 87(2):373-379, 373-379,(2008). (2008). 31. 31. Weintroub, Weintroub,S.S.Reddi, Reddi,A.H., A.H.,Influence Influenceofofirradiation irradiationon onthe the osteoinductive osteoinductivepotential potentialofofdemineralized demineralizedbone bonematrix. matrix.Calcif. Calcif. Tissue TissueInt. Int.42(4): 42(4):255-260, 255-260,(1998). (1998). Puros Puros®®isisaaregistered registeredtrademark trademarkofofZimmer, Zimmer,Inc. Inc.ororitsitsaffiliates. affiliates. Cancelle CancelleSP SP™™isisaatrademark trademarkofofRTI RTIBiologics, Biologics,Inc. Inc. 14. 14. Lacroix, Lacroix,P., P.,Recent Recentinvestigations investigationson onthe thegrowth growthofofbone. bone. Nature, Nature,156: 156:576-577, 576-577,(1945). (1945). 15. 15. Reddi, Reddi,A.H., A.H.,Huggins, Huggins,C.B., C.B.,Biochemical Biochemicalsequences sequencesininthe the transformation transformationofofnormal normalfibroblasts fibroblastsininadolescent adolescentrats. rats.Natl. Natl. Acad. Acad.Sci., Sci.,USA., USA.,69: 69:1601-1605, 1601-1605,(1972). (1972). 16. 16. Reddi, Reddi,A.H., A.H.,Cell Cellbiology biologyand andbiochemistry biochemistryofofendochondral endochondral bone bonedevelopment. development. Collagen CollagenRel. Rel.Res.1: Res.1:209-226, 209-226,(1981). (1981). 17. 17. Covey, Covey,D., D.,Albright, Albright,J.,J.,Clinical Clinicalinduction inductionofofbone bonerepair repairwith with demineralized demineralizedbone bonematrix matrixororaabone bonemorphogenetic morphogeneticprotein. protein. Ortho. Ortho.Rev., Rev.,18: 18:857-863, 857-863,(1989). (1989). 18. 18. Lee, Lee,K.J.H., K.J.H.,Roper, Roper,J.G., J.G.,Wang, Wang,J.C., J.C.,Demineralized Demineralizedbone bone matrix matrixand andspinal spinalarthrodesis. arthrodesis.The TheSpine SpineJ.,J., 5:5:217S-223S, 217S-223S, (2005). (2005). 19. 19. Reddi, Reddi,A., A.,Initiation Initiationofoffracture fracturerepair repairby bybone bonemorphogenetic morphogenetic proteins. proteins.Clin. Clin.Orthop. Orthop.Rel. Rel.Res,. Res,.355S:,66-72, 355S:,66-72,(1998). (1998). 20. 20. Landesman, Landesman,R., R.,Reddi, Reddi,A. A.H., H.,Chemotaxis Chemotaxisofofmuscle musclederived derived mesenchymal mesenchymalcells cellstotobone boneinductive inductiveproteins proteinsofofrat. rat. Calcif. Calcif. Tissue TissueInt., Int.,39: 39:259-262, 259-262,(1986). (1986). ©2009 Zimmer Dental, Inc. All rights reserved. ©2009 ©2009Zimmer ZimmerInc. Inc.All Allrights rightsreserved reserved 6 Osteoinductivity of Puros® DBM Putty in Athymic Rat Model Steven T. Moore and Ronald R. Cobb Introduction: Commercially available bone paste products consist of formulations that combine demineralized bone matrix (DBM) with a carrier material. RTI Biologics, Inc. (RTI) currently markets a series of allograft DBM paste products that are produced using single donors and a porcine-derived carrier. Recent efforts have focused on generating a 100% human-derived DBM paste product made from a mixture of two types of DBM from different stages in the manufacturing process. Stage 1 DBM (DBM-1) is a demineralized bone matrix powder that preserves the osteoinductive potential of the bone. Stage 2 DBM (DBM-2) is a completely demineralized bone matrix that when mixed with DBM-1 gives the final product a “putty-like” consistency. This ready-to-use product is stored hydrated at room temperature in a one stage delivery system and is marketed as Puros DBM Putty. Preliminary in vivo studies, using the rat ectopic pouch model, have shown that this new DBM product retains its osteoinductive properties. The athymic nude rat model described by Urist (1965) has been used to evaluate the osteoinductive potential and inflammatory response of DBM-based products for many years. To verify the osteoinductive potential of Puros DBM Putty (DBM-1 + DBM-2), samples of the finished product and DBM-1 alone, as a control, were implanted into the ectopic muscle pouches of athymic nude rats. After 28 days, the implants were removed and histology was reviewed to assess the osteoinductive potential as well as the inflammatory response of Puros DBM Putty (DBM-1 + DBM-2) and DBM-1 alone from the same donor. Materials and Methods: All human tissue used in this study was derived from consented cadaveric donations. DBM from a total of four different donors was selected for the current study, all had previously scored positive for osteoinductivity and did not exhibit significant inflammation by the QC athymic nude rat assay. The DBM-1 and DBM-2 for Puros DBM Putty were prepared from a single donor. All samples of Puros DBM Putty (DBM-1 + DBM-2) and DBM1 controls were irradiated at a dosage of 25-31 kGy. Biological activity was assessed (qualitatively and quantitatively) using the Urist (1965) athymic nude rat model. This in vivo model has been utilized by RTI to identify DBM with acceptable osteoinductivity for use in all paste products. After irradiation, each sample was implanted in triplicate in three separate rats with six samples per rat. The implants were extracted after 28 days and the samples were sent for histological preparation. The histological slides were scored for osteoinductivity and inflammatory responses in accordance with the scoring system of Edwards et al. (1998). In addition, bone maturity was scored in accordance with Katz et al. (2006). Results: Osteoinductivity and Bone Maturity: Osteoinductivity (OI) and bone maturity scores for Puros DBM Putty (DBM-1 + DBM-2), DBM-1 alone, and control treatment groups are presented in Table 1 below (also see Figures 1-5 for histological slides). As shown in Figures 1-5, addition of the carrier did not significantly affect the OI results of the Puros DBM Putty (DBM-1 + DBM-2) compared to the DBM-1 alone. In addition, there was no significant effect on the bone maturity scores in the Puros DBM Putty (DBM-1 + DBM-2) relative to the DBM-1 alone. Although the sample size was small (n = 4 donors), this data suggests that there is no significant effect of the Puros DBM carrier (DBM-2) on the osteoinductivity and bone maturity scores of DBM-1 alone. As expected, the OI scores for the negative control (deactivated DBM) samples were zero, as were the bone maturity scores. The average OI score for the positive control was 3.3 (± 0.58) with a bone maturity score of 9.0 (±0.00). Table 1. Mean ± Standard Deviation of Osteoinductivity (OI) and Bone Maturity Scores Tissue Treatment OI ± SD Maturity ± SD Donor 1 Puros DBM Putty 2.3 ± 0.58 DBM-1 Alone 2.0 ± 0.00 7.7 ± 0.58 7.7 ± 0.58 Donor 2 Puros DBM Putty 2.3 ± 0.58 DBM-1 Alone 2.3 ± 0.58 7.7 ± 0.58 8.3 ± 0.58 Donor 3 Puros DBM Putty 2.7 ± 0.58 DBM-1 Alone 3.3 ± 0.58 8.7 ± 0.58 9.0 ± 0.00 Donor 4 Puros DBM Putty 1.0 ± 0.00 DBM-1 Alone 1.7 ± 0.58 6.3 ± 1.20 7.7 ± 0.58 Controls Negative Control Positive Control 0.0 ± 0.00 9.0 ± 0.00 0.0 ± 0.00 3.3 ± 0.58 Inflammation: As shown in Table 2 below, Puros DBM Putty (DBM-1 + DBM-2) compared favorably to the DBM-1 alone with respect to inflammation scores. The addition of the DBM carrier (DBM-2) to the DBM-1 alone yielded inflammation scores that were in the acceptable range (inflammatory scores <_ 2). The deactivated DBM negative control generated an inflammatory score that would have resulted in rejection of the product (inflammatory scores > 2), a result which is consistent with results of past in vivo tests. 7 Table 2. Mean ± Standard Deviation of Inflammation Scores Tissue Treatment Inf. ± SD Donor 1 Puros DBM Putty 1.0 ± 0.00 DBM-1 Alone 1.3 ± 0.58 Donor 2 Puros DBM Putty 1.0 ± 0.00 DBM-1 Alone 1.3 ± 0.58 Donor 3 Puros DBM Putty 2.0 ± 0.00 DBM-1 Alone 1.3 ± 0.58 Donor 4 Puros DBM Putty 1.0 ± 0.00 DBM-1 Alone 1.0 ± 0.00 Controls Negative Control Positive Control 2.3 ± 0.58 1.0 ± 0.00 Histological Analyses: Histological analysis of the test implants failed to reveal qualitative differences between samples mixed with the DBM-2. Comparable remodeling features that are associated with new bone formation (cells associated with bone and marrow formation) were observed. Additionally there were no signs of inflammation associated with the Puros DBM Putty (DBM-1 + DBM-2) in the explants. Representative images of explants from matched donors with DBM-1 alone or Puros DBM Putty (DBM-1 + DBM-2) are shown in Figures 1-5. Conclusion: References: Edwards JT, Diegmann MH, Scarborough NL. Osteoinduction of human demineralized bone: characterization in a rat model. Clin Orthop Relat Res 1998(357):219-28. Katz, J. M., Diegl, E. C., Nataraj, C. Time course of DBM induced intermuscular endochondral ossification. Orthopedic Research Society Meeting (2006)abs.912. Katz, J.M., Nataraj, C., Jaw, R., Deigl, E., and Bursac, P. Demineralized bone matrix as an osteoinductive biomaterial and in vitro predictors of its biological potential. J. Biomed Mat Res: Part B - Applied Biomaterials, (submitted). Muthukumaran N, Ma S, Reddi AH. Dose-dependence of and threshold for optimal bone induction by collagenous bone matrix and osteogenin-enriched fraction. Coll Relat Res 1988;8(5):433-41. Sampath TK, Reddi AH. Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix. Biochem Biophys Res Commun 1984a;119(3):949-54. Sampath TK, Reddi AH. Importance of geometry of the extracellular matrix in endochondral bone differentiation. J Cell Biol 1984b;98(6):2192-7. Urist MR. Bone: formation by autoinduction. Science 1965;150(698):893-9. Zhang M, Powers RM, Jr., Wolfinbarger L, Jr. A quantitative assessment of osteoinductivity of human demineralized bone matrix. J Periodontol 1997;68(11):1076-84. A variety of qualitative features such as DBM particle size, calcium content, matrix integrity and the composition of the carrier are known to influence the osteoinductivity of bone paste products (Zhang et al., 1997; Mathukumaran et al., 1988; Sampath and Reddi, 1984a; Sampath and Reddi, 1984b). In the present study, the osteoinductivity of a bone paste product derived from 100% human material, Puros DBM Putty, was investigated in an in vivo rat ectopic pouch model. Recent findings demonstrate that this form of testing is the most reliable in terms of accurately determining the osteoinductive and inflammatory properties of bone paste products (Katz et al., submitted). The data clearly indicate that the DBM-2 (DBM carrier) has no significant dilutive effect on the osteoinductivity of DBM-1 alone as measured using the athymic nude rat model. Histological evaluation of the test samples also demonstrated that Puros DBM Putty (DBM-1 + DBM-2) had essentially the same remodeling features associated with new bone formation as DBM-1 alone. In addition, this new DBM product did not cause an increase in inflammation. The results of this study clearly reveal that Puros DBM Putty (DBM-1 + DBM-2) has similar osteoinductivity and inflammatory characteristics to the DBM-1 alone. ©2008 RTI Biologics, Inc. (RTI) Reprinted by Zimmer, Inc. with permission of RTI. Puros is a registered trademark of Zimmer, Inc. or its affiliates 8 +H124971105014001/$090113A09Y 97-1105-014-00 2ML Printed in USA ©2009 Zimmer, Inc. A C A C A B C C B A A C A Figure 1. Donor 1, Puros DBM Putty (DBM-1 + DBM-2) Figure 2. Donor 1, DBM-1 Alone B C A B C A B C C C B C A Figure 3. Donor 3, Puros DBM Putty (DBM-1 + DBM-2) A Figure 4. Donor 3, DBM-1 Alone A= New Bone; B= DBM; C= Bone Marrow C B C C B Figure 5. Positive Control 9 Effect of Terminal Gamma Sterilization Effect of Terminal Gamma on Osteoinductivity Effect of Terminal Gamma Sterilization on Osteoinductivity Effect of Terminal Gamma Sterilization Effect of Terminal Gamma Sterilization on Osteoinductivity on Osteoinductivity Chandra Nataraj, BVSc, PhD; Elizabeth Silveira, BS; James Clark, MS; Joan Yonchek, BS, HT; and James Kirk, PhD on Osteoinductivity (RTI Biologics, Inc., Alachua, FL) Chandra Nataraj, BVSc, PhD; Elizabeth Silveira, BS; James Chandra Nataraj, BVSc, PhD; Elizabeth Silveira, BS; James Clark, MS; Joan Yonchek, BS, HT; and James Kirk, PhD (RTI Biologics, Inc., Alachua, FL) Methods Introduction (RTI Biologics, Inc.,BVSc, Alachua, Chandra Nataraj, PhD;FL) Elizabeth Silveira, BS; James Clark, MS; Joan Yonchek, BS, HT; and James Kirk, PhD In order to ensure that gamma irradiation at sterilization doses (2.5RTI Biologics, Inc. (RTI) currently produces allograft demineralized Introduction (RTI Biologics, Inc., Alachua, FL) 3.1 Mrad) does not compromise RTI the biological of DBM paste produces allograft bone matrix (DBM) paste products that are manufactured under strict Biologics,efficacy Inc. (RTI) currently Methods Introduction products, we assessed its effect bone on the osteoinductive property of aseptic conditions using single donors. All donors are subjected to matrix (DBM) paste products In order to ensure that gamma irradiation at sterilization doses (2.5-that are manufactu RTI Biologics, Inc. (RTI) currently produces allograft demineralized Methods Introduction demineralized bone matrix (DBM). A series of studies was performed rigorous screening, prior to processing, to ensure that no transmissible aseptic conditions using singlepaste donors. All donors a 3.1 Mrad)todoes notthat compromise the biological efficacy of DBM bone matrix (DBM) paste products that are manufactured under strict In order ensure gamma irradiation at sterilization doses (2.5RTI Biologics, (RTI) produces demineralized that compared the biological activity screening, of both precursor DBM pathogens are Inc. present in currently donor material. Inallograft addition, a terminal rigorous prior to processing, products, we assessed its effect on the osteoinductive property of to ensure that n aseptic conditions using single donors. All donors are subjected to 3.1 Mrad) notplus compromise the biological efficacy of DBM paste bone matrix (DBM) pastehas products that are manufactured underDBM strict powder anddoes DBM a porcine-derived collagen carrier (RTI Paste) sterilization procedure been introduced for all allograft pathogens are present in donor material. In additi demineralized bone matrix (DBM). A series of studies was performed rigorous screening, prior to processing, to ensure that no transmissible products, assessed itsirradiation. effect on the osteoinductive property of aseptic conditions using single donors. All donors are subjected to before andweafter gamma Biological activity was assessed paste products. sterilization procedure has been introduced for all a that compared thematrix biological activity ofofboth precursor DBM pathogens are present material. In addition, a terminal demineralized bone (DBM). A series studies was performed rigorous screening, priorintodonor processing, to ensure that no transmissible (qualitatively and quantitatively)paste using the Urist athymic nude rat products. sterilization procedure has been introduced for all allograft DBM powder and DBM plus a porcine-derived collagen carrier (RTI Paste) 8 that compared themodel biological activity of toboth precursor DBM pathogens are presentisinandonor material. Gamma irradiation accepted meansInofaddition, achievinga terminal model. This in vivo is utilized by RTI identify DBM powder before and gamma Biological activity was assessed paste products. powder andafter DBM plus a irradiation. porcine-derived collagen carrier (RTI Paste) sterilization procedure introduced forbiologics. all allograftPrevious DBM sterilization in a varietyhas of been products including with acceptable osteoinductivity for use in all DBM paste products. Gamma irradiation is an nude accepted (qualitatively andgamma quantitatively) using the Urist athymic rat means of achie before and after irradiation. Biological activity was assessed paste products. reports indicate the use of this method for decades in the allograft 88 sterilization in a variety of products including biolo Gamma irradiation is an accepted means of achieving terminal model. This in vivo model is utilized by RTI to identify DBM powder (qualitatively and quantitatively) using the Urist athymic nude rat transplant arena.1-3 Gamma irradiation at relatively low doses (~1 Results reports indicate thepaste use products. of this method for decades i sterilization in a variety of accepted products means including biologics. Previous 8 with acceptable osteoinductivity for use in all DBM Gamma irradiation is an of achieving terminal model. This in vivo model is utilized byand RTI to Paste identify DBMprepared powder Mrad) is capable of killing most classes of microorganisms, with small 1-3 samples In a comparative study, DBM powder RTI Gamma irradiation at relatively transplant arena. reports indicateathe use of method for decades in the Previous allograft sterilization variety of this products biologics. with acceptable osteoinductivity for use in all preparations DBM paste products. viruses beinginthe exception. Sterility including by irradiation is accomplished across different donors were tested. Sample were either 1-3 Mrad) is capable of killing most classes of microorgani transplant arena. Gamma irradiation at relatively low doses (~1 Results reports indicate the use ofofthis method decades in therendering allograft primarily by the alteration nucleic acidsfor (RNA and DNA) irradiated in an environmentally controlled method or left untreated, viruses being the exception. Sterility by irradiation i 1-3 killing most classes of microorganisms, with small Mrad) is capable of In a comparative study, DBM powder and RTI Paste samples prepared transplant arena. Gamma irradiation at relatively low doses (~1 Results life forms incapable of reproducing. A secondary effect of irradiation is after which they were implantedprimarily in abdominal muscle pouches ofacids (RNA and D by the alteration of nucleic viruses being the exception. Sterility by irradiation is accomplished Mrad) is capable killing mostWhile classesthese of microorganisms, with small across different donors were tested. Sample preparations were either In a comparative study, DBM powder and RTI Pastepost-implantation, samples prepared the generation of of free radicals. are capable of contributing athymic nude rats. Explants were retrieved 4-weeks life forms incapable of reproducing. A secondary effect primarily by the alteration of nucleic acids (RNA and DNA) rendering viruses being the exception. Sterility by irradiation is accomplished irradiated in an environmentally controlled method or left untreated, across different donors were tested. Sample preparations were either to the sterilization process, free radicals have also been shown to alter processed, and evaluated histologically for evidence of bone formation the generation of free radicals. While these are capable life forms incapable of reproducing. A secondary effect of irradiation is after which they were implanted in abdominal muscle pouches of 4-7 rendering primarily by the and alteration of nucleic (RNA and DNA) irradiated in an environmentally controlled method or left untreated, the mechanical biological qualityacids of bone allografts. and inflammation. For data analysis, thesterilization overall osteoinductivity (OI) have also been to the process, free radicals the generation of free radicals. While these are capable of contributing life forms incapable of reproducing. A secondary effect of irradiation is athymic nudethey rats.were Explants were retrieved 4-weeksmuscle post-implantation, after implanted inmechanical abdominal pouches of of bone allograft score which obtained for the pre-gamma irradiation group used as quality a base the andwas biological Previous studies shown mixed results with respect to thetoeffects to the sterilization process, free radicals have also beenofshown alter the generation of have free radicals. While these are capable contributing processed, and evaluated histologically for evidence of bone formation athymic nudeand rats.scores Explants were retrieved 4-weeks post-implantation, 4-7 line of 100% given to their irradiated counterparts expressed 4-7 of the gamma irradiation on thefree biological properties the mechanical andprocess, biological quality ofand bonebiomechanical allografts. and inflammation. For data analysis, the (OI) results with respec to sterilization radicals have also been shown to alter Previous studiesosteoinductivity have shown mixed processed, evaluated histologically foroverall evidence of bone formation as a percentand thereof. 4-7 4-7 In and general, gamma irradiation ofallografts. bone allografts at low of bone. the mechanical biological quality of bone score obtained for the pre-gamma irradiation group was used as a base and biomechan of gamma irradiation on the biological and inflammation. For data analysis, the overall osteoinductivity (OI) Previous studies have shown mixed results with respect to the effects temperatures appears to preserve both mechanical and biological 4-7 line of 100% and scores given to their irradiated counterparts expressed In general, gamma irradiation of bone al of bone. score for biological the pre-gamma group was used as(DBM a base Table 1obtained depicts the activityirradiation seen for each implant group of gammastudies irradiation the biological and with biomechanical properties Previous have on shown mixed results respect to the effects properties. as a percent thereof. 4-7 In the present study, the effects of gamma sterilization on 4-7 temperatures appears to preserve both mechanical line of 100% and scores given to their irradiated counterparts expressed of bone. In general, gamma irradiation of bone allografts at low powder or RTI Paste) before and after gamma irradiation. of irradiationofonallograft the biological andproducts biomechanical properties thegamma osteoinductivity bone paste were investigated properties. In the present study, the effects of gamma as a percent thereof. 4-7 temperatures appears to preserve both mechanical and biological In general, gammaof irradiation of bone allografts at low of Table 1 depictsanalysis the biological activity seen forfailed each implant group (DBM in bone. a rat ectopic pouch model bone formation. the osteoinductivity of allograft bone paste products we Histological of the test implants to reveal qualitative properties. In the present study, the effects of gamma sterilization on temperatures appears to preserve both mechanical and biological powder orinRTI Paste) beforeactivity andirradiation. after gamma irradiation. Table 1 depicts the biological seen for each implant group (DBM changes samples following Comparable remodeling in a rat ectopic pouch model of bone formation. the osteoinductivity of allograft bone paste products were investigated properties. In the present study, the effects of gamma sterilization on powder or RTI Paste) beforewith and new afterbone gamma irradiation. features that are associated formation (cells associated in a rat ectopic pouch model of bone formation. the osteoinductivity of allograft bone paste products were investigated Histological analysis of the test implants failed to reveal qualitative with bone and marrow formation) were observed. Additionally there in a rat ectopic pouch model of bone formation. changes in samples following irradiation. Comparable remodeling Histological of the test[usually implants failed towith reveal were no signsanalysis of inflammation associated the qualitative generation features that are associated with9 irradiation. new bone formation (cellsremodeling associated changes in samples following Comparable of free radical associated toxins ] in the explants as well. Representative with bone and marrow formation) were observed. Additionally there features thatand are high associated newofbone formation (cells associated low power powerwith images explants from matched donors were no signs of inflammation [usually associated with the generation with observed. beforebone and and aftermarrow gamma formation) irradiation were are shown here.Additionally (Figure 1) there of freenoradical toxins99[usually ] in the explants as well. were signs associated of inflammation associated with Representative the generation 9 lowfree power andassociated high power images of explants from donors of radical toxins ] in the explants as well.matched Representative before and after gamma irradiation are shown here. (Figure 1) low power and high power images of explants from matched donors before and after gamma irradiation are shown here. (Figure 1) Table 1. Table. 1 Table 1. Table 1. 1 Sample Type No. Implants Pre-Gamma OI Post-Gamma OI P value (t-test) Sample No. Pre-Gamma Post-Gamma P0.3** value RTI Paste 24 100 ± 7.8 92.5 ± 8.8 Type Implants OI OI (t-test) Sample No. Pre-Gamma Post-Gamma P value 2 DBM Powder 24 100 ± 7.3 103 ±- 4.1 0.39** Type Implants OI OI (t-test) 1 RTI Paste 24 100 ± 7.8 92.5 ± 8.8 0.3** ** W1henOI OIRTI scores were compared between the groups using the t-test, statistically significant **When scores were compared between the groups using the t-test, statistically significant differences Paste 24 100 ± 7.8 92.5 ± 8.8 0.3** 2 NOTDBM Powder 24 100 ± 7.3 103 ±- 4.1 0.39** were seenwere between groups. differences NOTtheseen between the groups. 2 DBM Powder 24 100 ± 7.3 103 ±- 4.1 0.39** **When OI scores were compared between the groups using the t-test, statistically significant differences were NOT the groups. **When OIseen scoresbetween were compared between the groups using the t-test, statistically significant differences were NOT seen between the groups. 10 Table 1. Sample Type No. Implants Pre-Gamm OI 1 RTI Paste 24 100 ± 7.8 2 DBM Powder 24 100 ± 7.3 **When OI scores were compared between the groups using were NOT seen between the groups. irradiation on the osteoinductivity and osteoconductivity of DBM.5,10 In the present study, a direct comparison of non-gamma irradiated and gamma irradiated (2.5-3.1 Mrad) DBM was conducted in an in vivo rat ectopic pouch model. The data clearly indicate that gamma irradiation has no significant effect on the osteoinductivity of DBM powder or DBM-based products with porcine-derived collagen carrier Conclusion when tested in this model. Histological evaluation the test Previous studies have suggested a possible negativeofeffect of samples gamma Conclusion also demonstrated that gamma irradiation had no negative effects 5,10 irradiation on the osteoinductivity and osteoconductivity of DBM. Previous studies have suggested a possible of gamma on the remodeling features associated with negative newnon-gamma boneeffect formation. In In the present study, a direct comparison of irradiated irradiation ongamma the osteoinductivity and osteoconductivity ofincrease DBM.5,10 addition, the irradiation of the DBM did not cause an andthe gamma irradiated Mrad) DBM conductedirradiated in an in in In present study, a(2.5-3.1 directtests comparison oflotwas non-gamma inflammation. RTI routinely each donor material in this animal vivo rat ectopic pouch model. The data clearly indicate that gamma and gamma irradiated (2.5-3.1 Mrad) DBM was conducted in an in model to ensure thatsignificant gamma irradiation, as well as our other processing irradiation has no effect on the osteoinductivity ofgamma DBM vivo rat ectopic pouch model. The data clearly indicate thatpotential protocols, does not have a negative effect on the osteoinductive powder or DBM-based products with porcine-derived collagen carrier irradiation has no significant effect on the osteoinductivity of DBM of its DBM paste when tested in thisproducts. model. Histological evaluation of the test samples powder or DBM-based products with porcine-derived collagen carrier also demonstrated that gamma irradiation had no negative effects when tested in this model. Histological evaluation of the test samples on the remodeling features associated with new bone formation. In also demonstrated that gamma irradiation had no negative effects addition, the gamma irradiation of the DBM did not cause an increase in on the remodeling features associated with new bone formation. In inflammation. RTI routinely tests each donor lot material in this animal addition, the gamma irradiation of the DBM did not cause an increase in model to ensureRTI thatroutinely gamma irradiation, as well our other processing inflammation. tests each donor lotasmaterial in this animal protocols, does not have a negative effect on the osteoinductive potential model to ensure that gamma irradiation, as well as our other processing of its DBM paste protocols, does notproducts. have a negative effect on the osteoinductive potential of its DBM paste products. Pre-Gamma (Low Mag) Pre-Gamma (Low Mag) Pre-Gamma (Low Mag) Pre-Gamma (Low Mag) Post-Gamma (Low Mag) References 1. Devries, P.H., Badgley, C.E., Hartman, J.T. Radiation sterilization of homogenous bone transplants using radioactive cobalt. J. Bone Joint Surgery. 40:187-203. 1958. 2. Zasacki, W. The efficacy of application of lyophilized, radiation-sterilized bone graft in orthopedic surgery. Clin. Orthop. 272:82-87. 1991 3. Goclawska, D.A., Ostrowski, K., Stachowicz, W., Michalik, J., Grzesik, W. Effect of radiation sterilization on the osteoinductive properties and the rate References of remodeling of bone implants preserved by lyophilization and deep-freezing. 1. Clin. Devries, P.H., Badgley, C.E.,1991. Hartman, J.T. Radiation sterilization of homogenous Orthop. 272:30-37. References bone transplants using radioactive cobalt. of J. Bone Joint on Surgery. 40:187-203. 4. Devries, Wientroub, and Reddi, Influence irradiation the of osteoinductive 1. P.H.,S.,Badgley, C.E.,A.H. Hartman, J.T. Radiation sterilization homogenous 1958. of demineralized bone matrix. Calcif. Tissue Int. 42(4): 255-60. potential bone transplants using radioactive cobalt. J. Bone Joint Surgery. 40:187-203. 2. 1988. Zasacki, W. The efficacy of application of lyophilized, radiation-sterilized bone 1958. graft in orthopedic surgery.Puhlmann, Clin. Orthop. Kessler, 272:82-87. 1991 5. Hallfeldt, K.K., S., Shweiberer, L. Sterilization 2. Zasacki, W. TheStutzle, efficacyH., of applicationM., of lyophilized, radiation-sterilized bone 3. of Goclawska, D.A., Ostrowski, K., Stachowicz, W., Michalik, J., sterilization Grzesik, W. partially deminerallized bone Orthop. matrix: 272:82-87. the effects of different graft in orthopedic surgery. Clin. 1991 Effect of radiation sterilization on the osteoinductive properties and the rate techniques onD.A., osteogenic properties. J. Surg. Res.W., 59(5):614-20. 3. Goclawska, Ostrowski, K., Stachowicz, Michalik, J.,1995. Grzesik, W. of remodeling bone implants preserved by lyophilization and deep-freezing. 6. Effect Anderson, M.J.,ofKeyak, J.H., Skinner, Compressive mechanical of radiation sterilization on theH.B. osteoinductive properties andproperties the rate Clin. Orthop. 272:30-37. 1991. of human cancellous afterpreserved gamma irradiation. J. Boneand Joint Surg. Am. of remodeling of bonebone implants by lyophilization deep-freezing. 4. 74(5):747-52. Wientroub, S.,1992. and Reddi, A.H. Influence of irradiation on the osteoinductive Clin. Orthop. 272:30-37. 1991. potential of demineralized bone matrix. Calcif. Tissue Int. 42(4): 255-60. 7. Wientroub, Hamer, A.J.,S.,Stocley, I., Elson, Changes in allograft irradiated at 4. and Reddi, A.H. R.A. Influence of irradiation on bone the osteoinductive 1988. temperatures. J. Bone Joint Surg. Br. 81(2):342-44. 1999. different potential of demineralized bone matrix. Calcif. Tissue Int. 42(4): 255-60. 5. Urist, Hallfeldt, K.K., Stutzle, H., Puhlmann, M., Kessler, S., Shweiberer, L. Sterilization 8. 1988. M.R. Bone: Formation by autoinduction. Science. 150:893-99. 1965. of partially deminerallized bone theD. effects of different sterilization 9. Moreau, Gallois, Basle, M., matrix: Chappard, Gamma irradiation of human 5. Hallfeldt,M., K.K., Stutzle,Y.,H., Puhlmann, M., Kessler, S., Shweiberer, L. Sterilization techniques on osteogenic properties. J. Surg. Res. 59(5):614-20. 1995. bone allografts alters medullary lipids and releases toxic compounds osteoblast of partially deminerallized bone matrix: the effects of differentfor sterilization 6. like Anderson, M.J., Keyak,21:369-76. J.H., Skinner, H.B. Compressive mechanical properties cells. Biomaterials. techniques on osteogenic properties.2000. J. Surg. Res. 59(5):614-20. 1995. of human cancellous bone after gamma irradiation. J. Bone Joint Surg. Am. 10. Anderson, Goclawska, D.A.,Keyak, Kaminski, The effect radiation-induced degradation of 6. M.J., J.H., A. Skinner, H.B.ofCompressive mechanical properties 74(5):747-52. 1992. collagen oncancellous osteoinductive of bone allografts.J.Abstract (IE-7)Surg. presented of human boneproperties after gamma irradiation. Bone Joint Am. 7. at Hamer, A.J., Stocley, I., Elson, R.A.Banks, Changes allograft bone Boston, irradiated at the American Association of Tissue 26th in Annual Meeting, MA. 74(5):747-52. 1992. different temperatures. J. Bone Joint Surg. Br. 81(2):342-44. 1999. 7. 2002. Hamer, A.J., Stocley, I., Elson, R.A. Changes in allograft bone irradiated at 8. Urist, M.R. Bone: Formation by autoinduction. Science. 150:893-99. 1965. different temperatures. J. Bone Joint Surg. Br. 81(2):342-44. 1999. 9. Moreau, M., Gallois, Y., Basle, M., Chappard, D. Gamma irradiation of human 8. Urist, M.R. Bone: Formation by autoinduction. Science. 150:893-99. 1965. bone allografts alters medullary lipids and releases toxic compounds for osteoblast 9. Moreau, M., Gallois, Y., Basle, M., Chappard, D. Gamma irradiation of human like cells. Biomaterials. 21:369-76. 2000. bone allografts alters medullary lipids and releases toxic compounds for osteoblast 10. Goclawska, D.A., Kaminski, A. The effect of radiation-induced degradation of like cells. Biomaterials. 21:369-76. 2000. collagen on osteoinductive properties of bone allografts. Abstract (IE-7) presented 10. Goclawska, D.A., Kaminski, A. The effect of radiation-induced degradation of at the American Association of Tissue Banks, 26th Annual Meeting, Boston, MA. collagen on osteoinductive properties of bone allografts. Abstract (IE-7) presented 2002. at the American Association of Tissue Banks, 26th Annual Meeting, Boston, MA. 2002. ©2008 by RTI Biologics, Inc. (RTI) Reprinted by Zimmer, Inc. with permission of RTI. ©2008 by RTI Biologics, Inc. (RTI) Reprinted by Zimmer, Inc. with permission of RTI. ©2008 by RTI Biologics, Inc. (RTI) Reprinted by Zimmer, Inc. with permission of RTI. Post-Gamma (Low Mag) Post-Gamma (Low Mag) Post-Gamma (Low Mag) Pre-Gamma (High Mag) Pre-Gamma (High Mag) Pre-Gamma (High Mag) Pre-Gamma (High Mag) Post-Gamma (High Mag) Figure 1 Both low and high magnification images of RTI Paste explants at 4 weeks, stained with Hematoxylin-Eosin showing comparable cellular events associated with new bone formation, without an inflammatory Post-Gamma (High Mag) response. Post-Gamma (High Mag) Figure 1 Figure 1 and high magnification images of RTI Paste Both low explants 4 weeks, stained with images Hematoxylin-Eosin Both lowat and high magnification of RTI Paste Post-Gamma (High Mag) showing comparable cellular events associated with explants at 4 weeks, stained with Hematoxylin-Eosin Figure 1. Both lowshowing and bone high magnification images new formation, without an associated inflammatory comparable cellular events with of RTI Paste explants at 4 weeks, stained with response. new bone formation, without an inflammatory Hematoxylin-Eosin showing comparable cellular response. events associated with new bone formation, without an inflammatory response. +H124971105015001/$090113A09Z 97-1105-015-00 2ML Printed in USA ©2009 Zimmer, Inc. 11 Comparative Handling Properties of Puros® Demineralized Bone Matrix Putty Alan Adams, Hai Bo Wen, Rama Akella, and Ross Garrett Copyright 2009 by Zimmer, Inc. Introduction Demineralized bone matrix (DBM) is widely used as a bone void filler at sites that are intrinsically non-load bearing. Since dry DBM is difficult to deliver and contain at the surgical site, commercially available DBMs are constituted with a carrier to improve handling characteristics for surgeons. In most instances, these carriers are inert, non-bone derived viscous substances that help to hold the DBM particles together. Puros DBM Putty is a 100% human allograft putty product made from a mixture of two types of DBM from different stages in the manufacturing process from the same donor allograft material. The physical handling characteristics of a DBM product, such as the ease of extrusion from its container, the ability to resist migration from the implant site, and its flowability and viscosity, depend on the combined effects of DBM particles and carrier. The handling properties of Puros DBM Putty were compared with those of other commercially available DBMs by testing the ease of extrusion from their original packed containers, rheological and viscoelastic properties, and their ability to stay cohesive in a fluid environment. Figure 1. MCR101 Rheometer (Anton Paar) Results and Discussion Extrusion: Paste, Gel and Putty samples that were packaged in syringe-like dispensers were used to test the ease with which these products could be extruded from their dispensers. Grafton Putty and DynaGraft II DBMs were not used in this test since their original packing was in jars. All tested samples were extrudable from their containers and fell within the reported comfortable range for use in the operating room (4). Accell Connexus DBM required the maximum force (9.414 +/- 0.98lbs) while DBX Putty and Grafton Gel DBMs required the least force (1.460 +/- 0.81 and 1.461 +/- 0.43lbs respectively). Puros DBM Putty was easy to extrude and required 2.921 +/- 0.67lbs of force (Figure 2). Materials and Methods The extrusion test was conducted using a TA.XT2 Texture Analyzer (Stable MicroSystems) with a 5Kg load cell. The dispenser containing the DBM was placed into the custom–made fixture and the center of the probe was aligned in contact with the plunger. The contents of the dispenser were displaced by forward extrusion at 0.5mm/sec (1,2). The peak extrusion force was measured and data was expressed as mean +/- standard deviation (S.D.) from 5 samples. The cohesive property of the DBMs was tested by placing 0.5cc of each of the test samples in 10 ml of heparinized bovine plasma at 37oC in a humid reaction chamber. The samples were photographed just before the addition of the bovine plasma and thereafter at 2, 4, 6, 8, and 10 minutes. The viscoelastic properties of 2cc of the material were measured using a MCR101 Rheometer (Anton Paar) (Figure 1) at room temperature. The 25mm parallel plate geometry was lowered to a 3mm gap and oscillation measurements (10/sec at 10Hz) were made from 0.001 to 100% strain(3). The storage and loss moduli (G’ and G”), linear viscoelastic range (LVE) and complex viscosity were calculated using Rheoplus™ software.* 12 10 Extrusion Force (lbs) All human tissue used to make Puros DBM Putty was derived from consented cadaveric donations. Competitive DBM products included Accell Connexus® (Integra Life Sciences Corp.), DynaGraft® II (Integra Life Sciences Corp.), Grafton® Putty (Osteotech, Inc.), Grafton® Gel (Osteotech, Inc.), and DBX® Putty (Musculoskeletal Transplant Foundation), and were obtained from commercial sources.* 8 6 4 2 0 DBX Putty Grafton Gel Puros DBM PUROS Accell Connexus Acell Connexus Figure 2. Measurement of force required to extrude product from its container as a measure of ease of extrusion Cohesiveness in Wet Environment: In the clinic, DBM is implanted in a highly moist and irrigated environment. It thus becomes imperative that the graft resists quick structural disintegration in order to be effective. The cohesiveness test of the DBMs was conducted in a fluid (heparinized bovine plasma at 37oC) environment over 10 min. duration. A few DBM particles from Puros DBM Putty separated over the duration of the study while Grafton Putty and Grafton Gel DBMs disintegrated almost completely within the first 2 min. DynaGraft II and Accell Connexus DBMs, which contain a synthetic reverse phase medium known to harden at 37oC, maintained shape and integrity over the duration of the study. DBX Putty with sodium hyaluronate as the carrier also retained shape and integrity over the duration of the study. Puros DBM Putty was comparable to DynaGraft Putty, DBX Putty and Accell Connexus DBMs for up to 4 minutes (Figure 3), providing reasonable time for surgical site manipulation. After 4 min. in the fluid environment, Puros DBM Putty began to swell. In air DBM 2 min 4 min When plotted against the loss/storage modulus ratio (G”/G’) as in Figure 5, Puros DBM Putty shows favorable handling properties, being neither too flowable nor too thick, and without being too dry or stiff, thus allowing proper molding and shaping to fit a variety of bone void applications. 6 min 8 min 10 min Puros DBM PUROS Grafton Putty Grafton Gel Dynagraft II Putty DBX Figure 5. Flow characteristics of material. Storage and Loss modulus ratio as a function of complex viscosity of various DBMs. = Favorable Flow Characteristics Acell Accell Connexus Connexus Figure 3. Cohesiveness of DBMs in heparinized bovine plasma at 37oC Viscoelastic Properties of DBM: Complex viscosity, linear viscoelastic range, and storage and loss modulus measurements provide information on the relative stiffness and structural stability of a material (5). The effect of these variables on the handling characteristics of materials is shown in Figures 4 and 5. The linear viscoelastic range (LVE %) describes the structural stability of a material. When plotted against the loss/storage modulus ratio (G”/G’) as in Figure 4, Puros DBM Putty shows the highest structural stability, without being too dry, stiff, or flowable. The complex viscosity describes the flow characteristics of a material. Conclusion The critical factors that affect physical handling characteristics of DBM pastes, putties or gels are the ease with which the product can be extruded from its container, their ability to remain within a very moist graft site and the ease with which they can be shaped and molded to fill a defect of various sizes and shapes. Puros DBM Putty can be easily extruded from its dispenser without being too flowable. It also demonstrates highly favorable viscoelastic properties, making it easily moldable without being sticky or dry. The retained cohesiveness of Puros DBM Putty in a wet environment is comparable to or better than competitive DBMs. Comparing the Handling Properties of DBM Runny 0.40 References: Grafton Gel 1. Zimmer SOP Syringability 12_11_06 0.35 G”/G’ 0.30 DBM Putty 0.25 0.20 Puros DBM 0.15 Stiff 2. Jones, D.S., Woolfson, A.D., Brown, A.F. & O’Neill, M.J. Mucoadhesive, syringable drug delivery systems for controlled application of metronidazole to the periodontal pocket: In vitro release kinetics, syringability, mechanical and mucoadhesive properties. Journal of Controlled Release, 1997; 49: 71-79 0.10 0.2 Accell Connexus DynaGraft II Unstable 0.4 0.6 0.8 Structural Stability LVE (%) 1.0 Stable Figure 4. Relative stiffness and structural stability. Storage and Loss modulus ratio as a function of linear viscoelasticity of various DBMs. = Favorable Structural Stability 3. Zimmer SOP Rheology_01_09_06 4. Mathiowetz V., Kashman N., Volland G., Weber K., Dowe M., and Rogers S. Grip and pinch strength: Normative data for adults. Arch Phys Med Rehabil, 1985; 66: 69-72 5. Mezger, T. G.: The Rheology Handbook, for users of rotational and oscillatory rheometers. Vincentz: Hannover, 2002 * All trademarks and registered trademarks are the property of their respective owners. 97-1105-011-00 09907-T04 1ML Printed in USA ©2009 Zimmer, Inc. 13 Puros® Demineralized Bone Matrix Patient Safety Through Redundant Safeguards Introduction Every surgical procedure involves risk from multiple factors. The question is how to reduce that risk to an absolute minimum. At RTI Biologics, redundant safeguards - through three different stages - maximize tissue safety: (1) donor screening, (2) laboratory testing, and (3) tissue preparation, including RTI’s proprietary sterilization processes, validated to address potential disease transmission. Stage 1: Screening for Patient Safety After consent for donation is obtained, potential donors are screened for risk factors associated with infectious diseases and medical conditions that would rule out donation. Screening includes, but is not limited to: • Family/next-of-kin interview • Medical/hospital record review • Behavioral/lifestyle risk assessment • Medical examiner/coroner’s report (autopsy report, when available) • Laboratory, pathology and radiology reports Stage 2: Testing for Patient Safety An extensive panel of infectious disease tests is performed on each donor. The testing is done in a CLIA certified laboratory using test kits cleared, approved or licensed by the United States Food and Drug Administration (FDA) for donor testing. The results are subject to stringent acceptance criteria in order to release the donor tissue to the processing stage. Tests Performed • HCV Antibody • HBV Surface Antigen • HIV 1 & 2 Antibody • HBV Total Core Antibody • HTLV I & II Antibody • Syphilis • HIV-I/NAT • HCV/NAT Microbiological testing is used appropriately throughout the process to screen for potential contamination and to provide confirmation of tissue suitability for transplant. Microbiological Testing • Pre-processing culturing: Performed before processing begins to remove potentially unsuitable tissue • Environmental controls: Monitors cleanliness of processing environment The final determination of donor eligibility is made by RTI’s medical director—a licensed physician—utilizing all available, relevant screening and testing information. 97-1105-013-00 Rev.2 .25ML Printed in USA ©2008, 2010 Zimmer, Inc. 14 Stage 3: Validated Tissue Processing Demineralized Bone Products “The demineralization process inactivated infectious retrovirus in infected cortical bone, thereby preventing disease transmission.”* Sterilized Through the Cancelle™ SP DBM Sterilization Process The Cancelle SP process is a validated bone matrix sterilization process that inactivates potential pathogens through a combination of chemical treatments and gamma irradiation. The validation study used “model” viruses, closely related viruses having similar physical and chemical properties, in accordance with FDA guidance.** The validation study also included “challenge” viruses which test the ability of the demineralization process to inactivate the most resistant viruses as a measure of its overall viral inactivation capability. The Cancelle SP process is validated to inactivate the following model, relevant and challenge viruses: • Bovine Viral Diarrhea Virus (BVDV) Model • Human Immunodeficiency Virus (HIV) • Hepatitis C Virus (HCV) • Human T-lymphotropic Virus (HTLV) • Pseudorabies Virus (PrV) Model • Hepatitis B Virus (HBV) • Human Poliovirus (Polio-1) Challenge • Porcine Parvovirus (PPV) Challenge For Puros Putty, the gamma irradiation dose is delivered terminally to achieve a sterility assurance level of 10-6. Delivering Patient Safety RTI is focused on patient safety. Redundant safeguards provide the highest level of confidence that patients will receive safe, high-quality tissue; in fact, RTI has a proven combined record of more than two million implants distributed with zero incidence of allograft-associated infection. Not all companies can match the level of tissue safety offered through RTI. * Cheryl L. Swenson and Steven P. Arnoczky: Demineralization for Inactivation of Infectious Retrovirus in Systemically Infected Cortical Bone: In Vitro and in Vivo Experimental Studies; J. Bone Joint Surg. Am., Feb 2003; 85: 323 - 332. ** US Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER). 1998. “Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin.” Puros is a registered trademark of Zimmer, Inc. or its subsidiaries. Cancelle™ SP (DBM Sterilization Process) is a trademark of RTI Biologics, Inc. Osteoinductivity of Puros® DBM Putty in Athymic Rat Model Following a One Year Shelf-Life Study Steven T. Moore, Rasa Zhukauskas, and Ronald R. Cobb Introduction: The use of bone graft substitutes in orthopedic bone grafting procedures has increased dramatically in recent years. This is due in part to the wide range of materials, structures, and delivery systems that are available to be used. Additionally, bone graft substitutes often possess osteoconductivity and/or osteoinductivity. These characteristics make bone graft substitutes useful in augmenting the healing of bony defects caused by traumatic injury, tumor removal, abnormal skeletal development, cyst removal and prosthetic loosening. One widely used bone graft substitute material is demineralized bone matrix (DBM) constituted with a carrier. The DBM consists of insoluble collagen and non-collagenous proteins and has inherent osteoinductive and osteoconductive properties (Mulliken et al., 1984). While growth factors in DBM provide an osteoinductive effect, the collagen structure provides the osteoconductive effect. The carrier’s function is to facilitate handling characteristics and graft containment. The carrier keeps the DBM in place which allows the osteoinductive and osteoconductive nature of the DBM to facilitate bone regeneration to occur throughout a defect rather than simply at the edges (Mulliken et al., 1981). Although osteoinductive potential differentiates DBMs from synthetic bone graft substitutes, there are no known reports of osteoinductive potential over the entire shelf life period for commercially available DBMs. Puros DBM Putty is a commercially available product that is 100% human derived, consisting of stage 1 DBM (DBM-1) and stage 2 DBM (DBM-2). DBM-1 is a demineralized bone matrix powder that preserves the osteoinductive potential of the bone and DBM-2 is a further processed demineralized bone which, when mixed with DBM1, gives the final product a “putty-like” consistency. Puros DBM Putty has demonstrated osteoinductive potential in the athymic rat model described by Urist (1965). To verify the osteoinductive potential of Puros DBM Putty throughout the product shelf-life, samples of the finished product were implanted into the ectopic muscle pouches of athymic rats after 11, 30, 60, 210, 270, and 365 days of real-time aging respectively. After 28 days in the rat, the implants were removed and histology was reviewed to assess the osteoinductive potential as well as the inflammatory response. Materials and Methods: All human tissue used in this study was derived from consented cadaveric donations. DBM from a total of nine different donors that previously scored positive for osteoinductivity and did not exhibit significant inflammation by the QC athymic rat assay, was selected for the current study. Each Puros DBM Putty sample was prepared from a single donor (9 donors total). All samples and controls (inactivated DBM as negative control and inactivated DBM + BMP-2 as positive control) were gamma irradiated. Samples were stored at room temperature (15°-25°C) in RTI Biologics’ finished goods warehouse. At 11, 30, 60, 210, 270, and 365 day timepoints post-production (T0), individual samples were implanted into ectopic pouches of athymic rats. Biological activity was assessed (qualitatively and quantitatively) using the Urist (1965) athymic nude rat model. Puros DBM Putty samples were placed into the muscle pouches created in the ventral abdominal muscles of anesthetized athymic homozygous nude (nu/nu) rats. Seven athymic rats were used at each time point. Each rat received 6 implants. Twenty seven of the 42 implant sites at each time point were composed of Puros DBM Putty (3 samples from each of the 9 donors). The remaining 15 implant sites were composed of control DBM material. Puros DBM Putty implants and control DBM implants were randomized to eliminate any erroneous conclusion due to animal response variations. The implants were removed after 28 days. Explants were formalin fixed, decalcified, paraffin embedded, and cut into five micron sections. At least five sections were obtained per explant. The sections were stained with HematoxylinEosin. Osteoinductivity was analyzed using a semi-quantitative histological scoring system as described by Edwards, et al., (1998). In addition to the Edwards scoring system, bone maturity and the degree of inflammation for each section were also scored in accordance with Katz, et al., (2006). Results: Osteoinductivity and Bone Maturity: Previous studies have demonstrated that the 100% human Puros DBM Putty is osteoinductive (Moore et al., 2008). Osteoinductivity (OI) and bone maturity scores for the Puros DBM Putty stored for up to 365 days from one representative donor are presented in Table 1. Puros DBM Putty from each of the nine donors was shown to have potential for inducing new bone growth at each of the time points throughout this study (data not shown). Although there appears to be a trend towards a loss of osteoinductivity over time, these differences were not statistically significant. In addition, there were no significant differences in the bone maturity of each sample throughout the study (Table 1). Inflammation: Previous studies have shown that Puros DBM Putty compared favorably to DBM-1 alone with respect to generating bone growth and inflammatory response. Puros DBM Putty from a single donor, at the T0 + 30 time point only, had an unacceptable inflammation score. All other inflammation scores, including those of the DBM-1 from the deviating donor, were within the acceptable range (inflammation ≤ 2). The inflammatory response is scored on an increasing inflammation range of 1 to 4; a score of 2 is the highest acceptable inflammatory score. 15 Osteoinductivity Study Tissue Treatment Donor 3 Puros DBM Putty Negative Inactivated Control DBM-1 Positive Inactivated Control DBM-1 + BMP2 Aging Timepoints Test 11 Days 30 Days 60 Days 210 Days 270 Days 365 Days OI ± SD Mat. ± SD Inf. ± SD OI ± SD Mat. ± SD Inf. ± SD OI ± SD Mat. ± SD Inf. ± SD 3.0 ± 0.00 8.7 ± 0.58 1.0 ± 0.00 1.1 ± 1.95 2.6 ± 4.32 2.6 ± 0.69 4.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 3.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 1.0 ± 1.53 3.7 ± 4.64 2.0 ± 0.58 4.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 3.0 ± 0.00 8.7 ± 0.58 1.0 ± 0.00 1.0 ± 1.73 2.6 ± 4.39 1.6 ± 0.98 4.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 2.0 ± 0.00 7.7 ± 0.58 1.0 ± 0.00 1.1 ± 1.95 2.6 ± 4.39 1.9 ± 0.69 4.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 1.7 ± 0.58 8.0 ± 0.00 1.0 ± 0.00 1.1 ± 1.68 3.6 ± 4.50 2.0 ± 0.82 4.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 2.7 ± 0.58 7.7 ± 0.58 1.0 ± 0.00 1.1 ± 1.95 2.6 ± 4.39 2.3 ± 0.95 4.0 ± 0.00 9.0 ± 0.00 1.0 ± 0.00 Table 1. Mean ± Standard Deviation of Osteoinductivity (OI), Bone Maturity (Mat), and Inflammation (Inf) Scores Histological Analyses: Conclusion: Histological analyses of each sample yielded comparable remodeling features that are associated with new bone formation at all time points that were measured. Only Puros DBM Putty derived from Donor 8 showed no features of new bone formation at the T0 + 270 time point; however, at all previous time points and the T0 + 365 time point, this donor material did show evidence of new bone formation (data not shown). All other donor Puros DBM Putty samples demonstrated features of new bone formation. There were visible areas of marrow stromal matrix as well as pockets of osteoblasts and osteocytes. For each time point, representative images for Puros DBM Putty derived from a single donor are shown in Figure 1. Ideal bone graft substitutes should possess osteoinductive and osteoconductive properties, be biocompatible, and breakdown in concert with bony replacement. The osseous integration of a bone graft substitute depends on the activity of the surrounding bone cells and their precursors. Puros DBM Putty was shown to have osteoinductive potential in the athymic rat ectopic pouch model. The new bone formation was characterized by marrow stromal matrix and pockets of osteoblasts and osteocytes. Previous studies have shown that the athymic rat model is capable of responding to DBM implants from allogeneic and xenogeneic sources. This model distinguishes between active and inactive DBM implants. Recent studies have demonstrated that this model is the most dependable in terms of accurately determining the osteoinductive potential and inflammatory properties of bone paste products (Katz et al., 2008). Results Summary Puros DBM Putty samples, with a range of osteoinductive scores representing the final product, were used in this study to establish their osteoinductive potential and inflammatory responses over a 1 year period. The results showed the samples maintained osteoinductive potential with only a slight decrease in osteoinductivity as the year progressed. With the exception noted previously, all the donor materials induced limited inflammatory responses (≤2). The results show that Puros DBM Putty has the potential for inducing new bone growth for the duration of the 1 year shelf-life. 16 The data presented clearly supports that Puros DBM Putty maintains its osteoinductive properties for at least one year. Puros DBM Putty Samples from nine different donors were stored as final product and were tested at the given time points. Puros DBM Putty derived from each of the nine donors maintained its osteoinductive potential without eliciting a significant inflammatory response. T0 + 11 Days T0 + 30 Days T0 + 60 Days T0 + 210 Days T0 + 270 Days T0 + 365 Days Figure 1: Histology sections at each study time point for Puros DBM derived from a single donor 17 References Cited: ulliken JB, Kaban LB, Glowacki J. Induced osteogenesis--the M biological principle and clinical applications. J Surg Res 1984; 37 (6): 487. ulliken JB, Glowacki J, Kaban LB, Folkman J, Murray JE. Use M of demineralized allogeneic bone implants for the correction of maxillocraniofacial deformities. Ann Surg 1981; 194 (3): 366. Urist MR. Bone: formation by autoinduction. Science 1965; 150 (698): 893. Katz JM, Diegl, E. C., Nataraj, C. Time course of DBM induced intermuscular endochondral ossification, Orthopedic Research Society Meeting 2006: abs 912. Moore ST, Cobb RR. Osteoinductivity of Puros® DBM Putty in Athymic Rat Model. RTI Biologics Control Doc # 5330 (whitepaper), 2008. Urist MR, Strates BS. Bone formation in implants of partially and wholly demineralized bone matrix. Including observations on acetone-fixed intra and extracellular proteins. Clin Orthop Relat Res 1970; 71: 271. Katz JM, Nataraj C, Jaw R, Deigl E, Bursac P. Demineralized bone matrix as an osteoinductive biomaterial and in vitro predictors of its biological potential. J Biomed Mater Res B Appl Biomater 2009; 89 (b): 127. Puros is a registered trademark of Zimmer, Inc. or its subsidiaries. 5716 R0 09-23-09 © 2010 Zimmer Dental Inc. All rights reserved. 2090, Rev. 9/10. Puros Allografts are manufactured by RTI Biologics, Inc. and marketed by Zimmer Dental Inc. Please note that not all products and regenerative materials are registered or available in every country/region, and part numbers for Puros Allografts vary. Please check with a Zimmer Dental representative for availability and additional information. Edwards JT, Diegmann MH, Scarborough NL. Osteoinduction of human demineralized bone: characterization in a rat model. Clin Orthop Relat Res 1998; 357: 219.

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