Patent Abstract:
disclosed are methods for producing implantable bone compositions suitable for attaching stem cells thereto , characterized in that bone particles are contacted with an albumin comprising solution . said bone particles can be mineralized and / or lyophilized bone particles of animal or human origin . preferably the non - immunogenic albumin comprising solution is lyophilized onto said bone particles . the invention further concerns bone compositions suitable for use in graft implantation obtainable by said methods .

Detailed Description:
the term “ mineralized bone ” in the context of the present invention means that the bone is in its native form in a sense that it contains all of its mineral components and optionally it can contain its organic components as well . in other words the term “ mineralized bone ” is used to distinguish from demineralized bones in a sense that the mineralized bone is a non - demineralized bone . in accordance with the meaning as used herein , a bone composition is said to be “ substantially cleaned from organic components ” e . g . if it was prepared by the method described in the examples under the “ preparation of freeze - dried mineralized allograft ” section or by any other bone mineralization method comprising a washing step performed in an organic solvent and subsequent digestion in an acidic compound . a person skilled in the art would understand that there are numerous ways to produce an equivalent mineralized bone composition . coating of allografts with different proteins was surprisingly found to enhance the number of attached mscs on the surface of the graft , and also supplied the proliferation of cells . the allografts used as scaffolds were cleaned from almost all of the allogenic proteins of the bone in order to decrease the chance of any immunological complications in the host . the removed proteins , like low molecular weight proteins ( lmwps ) or bone morphogenetic protein ( bmp ) are important for the adherence and proliferation of the bone and osteoprogenitor cells ( 1 , 2 , 22 , 24 ). since these proteins had been removed , mscs could not attach onto the surface of the allograft itself , which mainly consisted of inorganic compounds , like hydroxide - apatite . present inventors have found that if the removed proteins are replaced with other proteins e . g . with fibronectin , albumin or collagen , preferably and most surprisingly with albumin , then these proteins facilitated the adherence of cells to the surface . furthermore this facilitated adherence was also observed by using a serum solution . this data shows that surprisingly not just the lmwps or bmps can support exclusively the attachment of bone progenitor cells , but other type of proteins as well . proliferation was observed on the coated grafts , except on those , which were coated with fibronectin or collagen type i alone . however , the combination of fibronectin with albumin and the combination of collagen type i with fibronectin provides good conditions for the proliferation of mscs . the best proliferation was observed when fcs was used as a coating material . proliferation was observed all along the investigation period on those grafts , which were coated with albumin alone or in combination with fibronectin and on those that contained fetal calf serum ( fcs ). our results show that albumin , fibronectin and the serum itself might be the most appropriate coating material for the bone allograft . these coating materials are easily available and supply the adherence and the proliferation of mscs . it has not escaped our attention that in a preferred embodiment of the invention the albumin , fibronectin , serum and of course the mscs could be autologous . a scaffold , which is free from the allogenic bone proteins and which contains autologous proteins of serum origin and autologous bone marrow derived mscs , could incorporate to the host faster than the conventionally used allo -, or autografts and enables an earlier recovery of the patient ( 9 , 10 , 11 , 17 ). although some mscs could adhere even on the surface of uncoated allografts , the mineralized bone surface that was cleaned from substantially all bmps could not provide good conditions for the survival of mscs ( fig1 ). viability of attached cells was investigated with alamar blue dye after 3 , 10 and 18 days . the color change of alamar blue indicated that the attached cells were alive on all of the grafts in the beginning of the experiment . alamar blue also indicated that attached cells were not alive , when their number had started to decrease on the grafts . the attached cells &# 39 ; viability was also investigated with calcein am fluorescent dye to confirm the results , which were obtained from the measurements with alamar blue . collagen type i could not supply the survival of mscs however it facilitated their adherence on the grafts ( fig2 a , b ). although fibronectin is commonly used to support the attachment of cells to different surfaces it facilitated neither adherence nor expansion of the mscs on mineralized bone allografts ( fig3 a , b ). on those grafts that contained fibronectin and collagen type i in combination , proliferation was observed in the beginning but the cells did not show metabolic activity after 10 days ( fig4 a , b ). the moisture content of the used protein compositions did not influence the results . these data show that although fibronectin and collagen type i are important bone structure proteins they themselves could not generate appropriate milieu for the survival of mscs ( fig5 ). surprisingly the presence of lyophilized albumin alone or in combination with other dried proteins of serum origin on grafts enhanced the number of attached mscs and they provided good conditions for the proliferation of cells all along the experimental period . when albumin or albumin - laden coating proteins were not subjected to freeze - drying on grafts after incubation in protein solution , the amount of cells decreased all along during the experimental period ( fig6 b , 7 b , 8 b ). onto those grafts that were coated with lyophilized albumin alone did not attach much more mscs than on untreated allografts . however we observed that there was a significant difference and this was that albumin facilitated the proliferation of mscs ( fig6 a ). combination of lyophilized albumin and fibronectin supplied the adherence and expansion of mscs as well ( fig7 a ). the highest proliferation rate in the last 8 days of the experiment was seen on those grafts that contained lyophilized fetal calf serum ( fig8 a ) ( data not showed on fig9 ). in conclusion our data show that coating of mineralized bone particles with serum and / or albumin not only supports stem cell attachment onto mineralized bone allografts , but also supports cell proliferation . therefore a reliable coating method was developed by the present inventors which coating method makes the surface of bone allografts , preferably mineralized and / or lyophilized bone allografts , appropriate to supply the attachment and survival of mscs . dulbecco &# 39 ; s modified eagle &# 39 ; s biochrom ag , germany medium fetal calf serum gibco , invitrogen , usa penicillin - streptomycin biochrom ag , germany l - glutamine biochrom ag , germany human albumin biotest hungária kft ., hungary 1000 ml solution contains : human plasma protein 200 g of which albumin is at least 96 % fibronectin sigma aldrich collagen type i pig collagen , 1 . 5 %, biom ′ up , france vybrant dyes molecular probes , invitrogen , usa calcein am molecular probes , invitrogen , usa alamar blue biosource , invitrogen , usa the percentages in the present description if otherwise not stated are always weight / weight percentages . bone marrow derived mesenchymal stem cells ( mscs ) were isolated from human bone marrow , and were expanded in dmem culture medium containing 10 % fetal calf serum , 2 mm l - glutamine , 100 u / ml penicillin and 100 μg / ml streptomycin . the bone marrow samples were obtained from young males and females aged 2 - 20 . only such tissues were used , that otherwise would have been discarded . semmelweis orthopedic clinic management committee , budapest , hungary , approved the use of these tissues . the bone marrow was taken into t75 flasks , and diluted with dmem culture medium . this mixture was stored in incubator at 37 ° c . in 5 % co 2 for 3 days . after the incubation time , the mscs adhered to the surface of the flask and the remnant components of bone marrow were eliminated by washing with pbs . the used cells were between 1 and 5 passages during the experiment . the identity of the adhered cells was confirmed by the presence of lineage - specific cell surface markers with flow citometry ( bd ® facscalibur , beckton dickinson , n . j ., usa ). haematopoetic linage - specific surface markers , like cd34 , cd45 and mesenchymal surface markers , like cd73 , cd90 , cd105 and cd166 were investigated . the used bones were obtained from cadavers or from surgical intervention . in the first step , the bones were washed in methanol for 4 hours . the methanol was changed continually during the procedure . after washing , the bones were digested in a solution that consisted of 0 . 1m phosphate buffer saline ( pbs ), 10 mm sodium - azide and 10 mm monoiodineacetic acid for 24 hours . subsequently , the bones were subjected to partial decalcification with 0 . 6 m hcl at room temperature for 4 to 6 hours and then they were washed in pbs . the produced mineralized bone structures ( matrices ) were sterilized in ethylene - dioxide at 27 ° c . and then they were freeze - dried ( lyophilized ) aseptically . process of the lyophilization : primer drying at 32 ° c ., 2 pa for 12 hours ; second drying at 32 ° c ., 0 pa for 12 hours . the mineralized allograft was frittered into cubic or round shape pieces . the superficies of the allograft pieces was one square centimeter and their height was 5 millimeters . thus prepared allografts were incubated in protein solution at + 4 ° c . overnight . the used protein solutions were 10 - 20 μg / ml fibronectin and 10 - 20 % albumin of human serum origin , furthermore fetal calf serum and 1 . 5 % collagen type i was derived from pig . the proteins were also used in combination , like fibronectin with albumin , fibronectin with collagen type i . these bones were used either directly for the “ without drying ” experiments , or subsequently , the incubated bones were lyophilized at 32 ° c ., at 1 pa for 24 hours . the coated allografts were subjected to uv irradiation for decontamination for 30 minutes . the seeded mscs were labeled with the fluorescent membrane dye vybrant did ( excitation / emission : 644 / 665 nm ) for 30 minutes at 37 ° c . did - labeled mscs were suspended in dmem culture medium , and dropped with pipette to the surface of the coated allografts . 60 . 000 cells / scaffold were used in all the experiments . the mscs were expanded on the allograft in vitro for 18 days . the seeded did - labeled msc &# 39 ; s proliferation was observed with confocal microscopy ( zeiss lsm 510 meta , carl zeiss , jena , germany ) on the coated allograft . individual areas of the graft were randomly selected , where the amount of the adhered cells were measured by fluorescence . the viability of the cells was investigated with alamar blue and calcein am dye . wistar rat ( 400 - 450 g ) femurs were harvested to use them as rat bone allografts . following the removal of bone marrow the rat femurs were milled to homogenous bone particles with a diameter of 1 mm . subsequently , the obtained rat bone particles were immersed into human albumin solution and incubated at + 4 ° c . overnight then the human albumin was freeze - dried onto the surface of said bone particles . the conditions of the freeze - drying : 32 ° c ., at 0 . 5 pa for 24 hours . wistar rat ( 400 - 450 g ) femurs were harvested to retrieve bone marrow derived stem cells . after the harvest of rat femurs their proximal and distal ends were dissected and the bone marrow was flushed into petri - dishes using dmem culture medium comprising 10 % fcs , 100 u / ml penicillin and 10 μg / ml streptomycin , 2 mm l - glutamine and 1 g / l glucose . the adhered cells at the bottom of petri - dishes were cultured at 37 ° c . in fully humidified atmosphere of 5 % co 2 ( standard culture conditions ) until the cells became confluent . subsequently , the cells were trypsinized and 80 . 000 cells were seeded on the surface of single rat bone allografts coated with freeze - dried human albumin . bone grafts prepared in this manner were stored under standard cell culture conditions for 7 days then the viability of attached cells was investigated with calcein am fluorescent dye using confocal microscope ( zeiss lsm 510 meta , carl zeiss , jena , germany ). we found that the rat bone allografts coated with freeze - dried human albumin provided adequate conditions for the attachment and survival of stem cells on the surface during the incubation period ( fig1 a , b ). the osseointegration ability of lyophilized bone grafts coated with freeze - dried human serum albumin was investigated in an animal model . in the first stage of the study a psuedoarthrosis model was developed . on wistar rats &# 39 ; ( 400 - 450 g ) femur a 2 - 3 mm wide transverse middiaphyseal osteotomy had been performed then the bone ends were fixed by plate and screws . polymethyl methacrylate ( pmma ) spacer was placed between the osteotomy sites and the periosteum was removed from the prepared femur to block natural bone healing . after 4 weeks the operation the pmma plate was removed and the osteotomy gap was left empty further 4 weeks . following 4 weeks post - operative period the animals were sacrificed and their femurs were harvested and subjected to μct examination to prove the development of a pseudarthrosis . in the second stage , the pseudoarthrosis model was prepared just as above detailed , but following the removal of the pmma spacers uncoated lyophilized bone allografts ( as control ) and coated with freeze - dried human albumin were implanted into the osteotomy gaps . 4 weeks after the implantation of bone grafts the animals were sacrificed and the harvested femurs were subjected to μct examination ( 25 ). new bone formation was not observed in the osteotomy gaps which were left empty following the removal of pmma spacer . this proves that the model is suitable for the investigation of the osseointegration ability of the bone grafts according to the present invention ( fig1 a , b ). the uncoated lyophilized bone allografts ( controls ) were not able to integrate into the site of bone defects ( fig1 c , d ). on the other hand , bony fusion can be detected 4 weeks after the implantation of the albumin lyophilized human bone graft . the cancellous bone between the osteotomy edges with a loose structure and wider holes refers to the remnants of the implanted graft . the x - ray shows heeling of the defect ( fig1 a , b ). 1 . mark e . bolander , gary balian . : use of demineralized bone matrix in the repair of segmental defects . u . s . pat . no . 4 , 743 , 259 , 10 may 1988 . 2 . mark e . bolander , gary balian . : use of demineralized bone matrix in the repair of segmental defects . u . s . pat . no . 4 , 902 , 296 , 20 feb . 1990 . 3 . barbara l . merboth , moon hae sunwoo , arthur a . gertzman . : allograft bone composition having gelatin binder . u . s . pat . no . 704 , 514 , 16 may 2006 . 4 . rust p a , kalsi p , briggs t w , cannon s r , blunn g w . : will mesenchymal stem cells differentiate into osteoblasts on allograft ? clin orthop relat res . 2007 april ; 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