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.

Description:
This is the National Phase of PCT/IB2009/050783, filed Feb. 26, 2009. 
     FIELD OF THE INVENTION 
     The invention relates to 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. In a preferred embodiment 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. 
     BACKGROUND OF THE INVENTION 
     Bone grafts are one of the most common transplanted tissues. Worldwide more than 2.2 million bone-grafting procedures are performed annually to repair bone defects in orthopedics, neurosurgery and dentistry (9, 10, 12, 13). Bone autografts and allografts are conventionally used to replace the bone defects. 
     Autografts are the gold standard to fill the bone defects, because they provide the fastest incorporation without immunological complications (15, 17). Few mature osteoblasts survive the transplantation however the number of precursor cells that stay alive is adequate. The osteogenic potential is derived from these precursor cells. It is well known in the art that there are severe limitations for the use of autografts these include the increased operative time, limited availability and significant morbidity related to blood loss, wound complications, local sensory loss and, most importantly, chronic pain (14, 16, 18, 19). 
     Allografts as an alternative approach offer similar characteristics with the exclusion of osteogenic cells (9, 10). There is not a standard rule related to the preparation of bone allografts thus several types of them are disclosed in publications, like fresh, fresh-frozen, freeze-dried or demineralized bone allografts (9, 10). Allografts possess osteoconductive and osteoinductive properties but the latter may not be recognized unless the graft is utilized in either morsellized or demineralized form. Complications associated with allografts include fracture, non-union, immunological complications and infection. 
     Urist and others (3, 20, 21, 22, 23, 24) demonstrated the effect of demineralized bone matrix or bone morphogenetic protein in bone induction. They also demonstrated that the active components of the demineralized bone matrix are the low molecular weight proteins (LMWPs). Bolander et al. (1, 2) coated the demineralized bone graft with additional low molecular weight proteins. This method could enhance osteoinductive potential of the graft. Nowadays, the demineralized allograft is still regarded as the most appropriate allogenic substitute for replace bone defects because it possesses osteoinductive and osteoconductive ability, however, its mechanical properties are not adequate (22). In addition manufacturing of demineralized bone matrix with constant high osteoinductive property is still a challenge. The mineralized or non demineralized bone allografts, for instance freeze-dried, fresh or fresh-frozen allografts possess good mechanical property but their osteoinductive capability is much poor compared with demineralized bone matrix. Rust et al. (4) demonstrated that mesenchymal stem cells (MSC) could differentiate into osteoblasts on the surface of a mineralized bone allograft, which contains the original proteins of normal bone. They found that MSCs could not differentiate on the surface of heat-treated allografts. This observation shows that particular bone proteins may play a key role in the adherence and commitment of MSCs. Booland et al. (5) proved that cells survive the impaction force on the mineralized allograft, which might be during the clinical use. Lewandrowski et al. (6) coated cortical bone grafts with biopolymers to support the adherence of periosteal derived bone cells on the graft&#39;s surface. 
     New biomaterials combined with osteogenic cells are now being developed as an alternative to bone grafts (11). The biomaterials are created to build up a three-dimensional scaffold to which cells can adhere, proliferate and differentiate into functional osteogenic cells (7, 9). Ore et al. (8) demonstrated that the enhanced differentiation of the human bone marrow derived cells on a 70% carbonated apatite, which has a composition similar to bone minerals. 
     Present inventors previously found that coating of spongy bone with proteins that are known to help cell adherence had not always helped cell adherence onto the bone. While coating with fibronectin increased the number of attached cells, coating with collagen did not help in increasing the number of attached cells. Moreover none of them supported the proliferation of the attached cells. 
     An optimal graft should have all the advantages of allo-, and autografts, including osteoinductive ability, good mechanical property and immunological compatibility with the host (10, 11). Furthermore preferably it is easily available and does not cause operative burden to harvest it, like autografts. 
     Our aim was to develop a reliable and safe coating method that ensures the attachment of stem cells onto the surface of mineralized bone allografts. In other words, the object of the present invention was to develop new methods that provide bone grafts that are compatible with the host and do not cause any immunologic complications while at the same time are easily available. 
     SUMMARY OF THE INVENTION 
     Present inventors solved the above problem according to a preferred embodiment of the invention by coating bone allografts of human or animal origin with freeze-dried human serum albumin. This preferred embodiment of the present invention provided excellent conditions for not just the attachment but also for the proliferation of stem cells e.g. mesenchymal stem cells (MSC). 
     The present invention relates to a method for producing implantable bone composition suitable for attaching stem cells thereto, characterized in that bone particles are contacted with a protein solution comprising albumin. While the concentration of albumin in said solution is not a strict limiting factor, preferably said albumin comprising solution is an albumin solution comprising 1 to 50% albumin, preferably 3 to 30% albumin, more preferably 5 to 25% albumin and most preferably approximately 10% albumin, wherein the percentages are weight/weight percentages. Preferably said albumin is human albumin and more preferably human albumin of serum origin. According to a preferred embodiment of the invention said bone particles are lyophilized bone particles and/or mineralized bone particles that are substantially cleaned from organic components. 
     In a preferred embodiment said albumin comprising solution is dried onto said mineralized bone composition and in a further preferred embodiment said drying is made by lyophilization. Said bone particles can be of human or animal origin. 
     Advantageously in the above method some of the ingredients of said albumin comprising solution is/are of human origin or is/are immunologically humanized and/or said albumin comprising solution is obtained from a patient in need of bone implantation. Preferably said albumin comprising solution comprises the serum of said patient or any fraction of said serum. 
     According to a further preferred embodiment of the present invention said albumin comprising solution preferably comprises recombinant albumin, wherein said albumin comprising solution optionally further comprises fibronectin or collagen or combination thereof. 
     Advantageously said protein solutions are free from components being potentially immunogenic to patients to whom said bone composition according to the invention is designed for. Methods for analyzing potential immunogenicity of a protein solution are well known in the art. 
     The invention concerns a method wherein the above methods further comprise a step of attaching cells capable of enhancing bone formation onto the surface of said bone particles. 
     In a preferred embodiment of the present invention in the above methods said cells capable of enhancing bone formation are obtained from a patient in need of a bone implantation, preferably said cells are mesenchymal stem cells (MSCs). 
     The invention further relates to a bone composition suitable for use in graft implantation, obtainable by the above methods of the invention. 
     The invention still further relates to the use of said compositions in therapy. 
     A graft prepared from a bone composition according to the present invention combines the beneficial properties of allo-, and autografts. Human mineralized bone allograft was used as a scaffold. A person skilled in the art would appreciate that instead of human mineralized bone allografts mineralized allografts of animal origin could be used equally well. The human allograft was cleaned from the bone original proteins. In this manner, the scaffold provides good mechanical stability to the graft; furthermore since it is mineralized it decreases the chance of any immunological complication. The scaffold&#39;s surface is coated with proteins of human origin, like fibronectin or albumin to supply the seeded adherence of human MSCs and proliferation. Surprisingly we found that coating with a serum solution (Fetal Calf Serum, FCS) helped the adherence of MSC to an even greater extent. The coating is necessary for cell viability on the allograft of the present invention and lyophilization of such proteins or protein solutions onto the bone enhanced the number of cells which were attached and supported their proliferation as well. In a preferred embodiment of the present invention the graft consists of materials of human origin, like autologous albumin and MSCs. This property could enable for the graft of the invention fast incorporation into the host without immunological complications, and could support an early recovery of patients. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the measured fluorescence, caused by the adhered cells on the surface of the untreated allografts 3, 10 and 18 days following the addition of MSCs. 
         FIG. 2A  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts onto which collagen type I was lyophilized, while  FIG. 2B  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts that were submersed into a collagen type I solution. 
         FIG. 3A  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts onto which fibronectin was lyophilized, while  FIG. 3B  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts that were submersed into a fibronectin solution. 
         FIG. 4A  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts onto which collagen type I and fibronectin was lyophilized, while  FIG. 4B  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts that were submersed into a collagen type I and fibronectin solution. 
         FIG. 5  shows the comparison of the measured fluorescence, caused by the albumin free coating materials. 
         FIG. 6A  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts onto which human albumin was lyophilized, while  FIG. 6B  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts that were submersed into a human albumin solution. 
         FIG. 7A  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts onto which human albumin and fibronectin was lyophilized, while  FIG. 7B  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts that were submersed into a human albumin and fibronectin solution. 
         FIG. 8A  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts onto which fetal calf serum was lyophilized, while  FIG. 8B  shows the measured fluorescence 3, 10 and 18 days following the addition of MSCs, caused by the adhered cells on the surface of allografts that were submersed into a fetal calf serum solution. 
         FIG. 9  shows the comparison of the measured fluorescence, caused by the albumin containing coating materials. 
         FIG. 10  A shows the surface of rat bone allograft coated with lyophilized human albumin which dose not contain adhered stem cells (act as control), while  FIG. 10  B shows the adhered, viable rat bone marrow derived stem cells on the surface of rat bone allograft coated with lyophilized human albumin. 
         FIG. 11A-B  show micro-CT snapshots of the inserted PMMA spacer between the dissected bone ends, while  FIG. 11C-D  show that the non-coated lyophilized human bone allograft did not facilitate new bone tissue formation therefore it could not integrate into the site of bone defect. 
         FIG. 12A-B  shows that a human bone graft coated with freeze-dried human albumin advanced new bone formation in the osteotomy gap following 4 weeks the implantation. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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 ( FIG. 1 ). 
     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 ( FIG. 2A , 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 ( FIG. 3A , 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 ( FIG. 4A , 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 ( FIG. 5 ). 
     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 ( FIGS. 6B ,  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 ( FIG. 6A ). Combination of lyophilized albumin and fibronectin supplied the adherence and expansion of MSCs as well ( FIG. 7A ). The highest proliferation rate in the last 8 days of the experiment was seen on those grafts that contained lyophilized fetal calf serum ( FIG. 8A ) (data not showed on  FIG. 9 ). 
     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. 
     EXAMPLES 
     Source of Used Materials 
                                     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.
 
     Example 1 
     Preparation and Investigation of Allografts 
     Isolation and culture of mesenchymal stem cells 
     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. 
     Characterization of MSCs 
     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. 
     Preparation of Freeze-Dried Mineralized Allograft 
     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. 
     Preparation of Coated Allograft 
     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. 
     Preparation of the Coated Allograft with Stem Cells 
     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. 
     Investigation of the seeded MSCs 
     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. 
     Example 2 
     Animal Bone Coated with Freeze-Dried Human Albumin 
     Preparation and Coating of Rat Bone Allografts 
     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. 
     Retrieval of Rat Bone Marrow Derived Stem Cells 
     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). 
     Results 
     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 ( FIG. 10A , B). 
     Example 3 
     Animal Model 
     Materials and Methods 
     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). 
     Results 
     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 ( FIG. 11A , B). 
     The uncoated lyophilized bone allografts (controls) were not able to integrate into the site of bone defects ( FIG. 11C , 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 ( FIG. 12A , B). 
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