in studying the problems associated with hematopoietic stem cell transplantation , the inventors developed an engineered bone marrow that recapitulates the cellular composition and the three - dimensional structure required for hsc replication and multilineage hematopoiesis , as in normal bone marrow . similarly , a methodology was established whereby the inventive composition , following transplantation in a mammal , increases the efficiency of hsc engraftment . an engineered bone marrow of the invention has both a cellular component and structural components that provide the three - dimensional structure associated with normal bone marrow . the cellular component includes any type of cell normally found within bone marrow (“ bone marrow cell ”) and , optionally , any type of hematopoietic stem cell ( hsc ). exemplary of the category of “ bone marrow cell ” are a stromal cell from bone morrow , a bone marrow stem cell , a fibroblast , an epithelial cell , an adipocyte , an osteocyte , an osteoblast , and a reticular cell . in this context , “ hematopoietic stem cell ” is an unspecialized cell that can undergo differentiation into a specific cell type in response to environmental signals . illustrative of a hematopoietic stem cell type is a bone marrow stem cell , a peripheral blood stem cell , and an umbilical cord stem cell , also known as a “ fetal stem cell .” bone marrow stem cells are the most common type of hsc . conventional methodology for isolating and maintaining bone marrow cells , prior to implantation , are described , for example , by thomas et al ., “ technique for human marrow grafting ,” blood 36 : 507 - 15 ( 1970 ), dexter et al ., “ regulation of hematopoietic stem cell proliferation in long term bone marrow cultures ,” biomedicine 27 : 344 - 49 ( 1977 ), and pittenger et al . “ multilineage potential of adult human mesenchymal stem cells ,” science 284 : 143 - 7 ( 1999 ). a peripheral blood stem cell ( pbsc ) is a stem cell that is found in the blood , albeit at a very low concentration . in order to collect pbscs , colony stimulating factor or growth factor is given to the donor to stimulate the bone marrow to produce more stem cells , which then are released into the blood stream . once they are in the blood , pbscs are collected in one or more sessions normally taking four to six hours each . in this procedure , called apheresis , the blood circulates through a machine called a cell separator , which removes the peripheral stem cells and returns the rest of the blood to the body . methodology for isolating and preparing peripheral blood stem cells are described , for instance , by majolino et al ., “ peripheral blood stem cells for allogeneic transplantation . recommendations from the gitmo 1996 ,” haematologica 81 : 529 - 32 ( 1996 ), novotny et al ., “ sustained decrease of peripheral lymphocytes after allogeneic blood stem cell apheresis ,” br . j . haematol . 100 : 695 - 7 ( 1998 ), and dini et al ., “ peripheral blood stem cell collection from g - csf - stimulated unrelated donors for second transplant ,” bone marrow transplant 22 ( suppl 5 ): s41 - 5 ( 1998 ). the final type of hsc is the umbilical cord stem cell . blood remaining in the placenta and umbilical cord of a newborn baby contains a large concentration of stem cells . the umbilical cord stem cells , instead of being discarded , may be harvested , and used for transplantation according to the methodology described by cairo et al ., “ placental and / or umbilical cord blood : an alternative source of hematopoietic stem cells for transplantation ,” blood 90 : 4665 - 4678 ( 1997 ), and kurtzberg et al ., “ placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients ,” n engl j med . 335 : 157 - 166 ( 1996 ). in a preferred embodiment , the engineered bone marrow is comprised of one or more bone marrow cell types that support the maintenance of hscs and their differentiation , especially in terms of multilineage hematopoiesis . examples of cell types involved in the differentiation of hscs include osteoblasts , adipocytes , endothelial cells , bone marrow fibroblasts , bone marrow reticular cells , and bone marrow stromal cells . in order to facilitate the engraftment of the hscs , the engineered bone marrow may be comprised of hscs and one or more other bone marrow cell types that have the same mhc haplotype . in a preferred embodiment , the engineered bone marrow contains the complete cellular component of bone marrow derived from a single individual . pursuant to the present invention , the structural components of an engineered bone marrow include ( i ) pulverized bone or a bone substitute and ( ii ) collagen or a collagen substitute . pulverized bone may be derived from any type of human or animal bone . sources of sterile bone can be obtained commercially from a variety of sources , including but not limited to the life link tissue bank ( tampa , fla . ), central florida tissue bank ( orlando , fla . ), and the pacific coast tissue bank ( los angeles , calif .). for this invention , any one of a number of different methods can be used to produce pulverize bone . these procedures are carried out under sterile conditions . in a preferred embodiment , the pulverized bone has a small particle size , such that the pulverized bone can be passed through an 18 - gauge needle , a size that allows the diffusion of nutrients to bone marrow cells . an alternative to pulverized bone in this context is a bone substitute . bone substitutes are described , for example , by marshal et al ., bone grafts , derivatives , and substitutes ( butterworth - heinemann lts ., 1994 ). calcium phosphates , such as hydroxyapatite , are generally considered materials of choice as bone substitutes . low - density hydroxyapatite is a preferred calcium phosphate bone substitute due to its ability to form a bond with collagen . examples of commercially available calcium phosphate bone substitutes are hapex ™, a product of smith & amp ; nephew richards , inc . ( memphis , tenn . ); durapatite ®, a product of acti - form ltd . ( markham , ontario ); and calcitite ®, which is manufactured by calciteck inc . ( san diego , calif .). in addition to calcium phosphates , there are a number of bioactive materials , such as glasses and ceramics , which may be used as bone substitutes . the common characteristic of these bioactive materials is that they are able to form a bond with bone or a soft tissue associated with bone such as collagen . in order to form a bond between tissues and the bioactive material , a layer of biologically active hydroxylcarbonate apatite ( hca ) must form on the bioactive material following implantation . in general , bioactive glasses and glass - ceramics require the combination of sio 2 , na 2 o , cao , and p 2 o 5 in specific proportions . there are three key compositional features to these bioactive glasses that distinguish them from traditional soda - lime - silica glasses : ( 1 ) they have less than 60 mole percent sio 2 , ( 2 ) they have a high na 2 o and cao content , and ( 3 ) they have high cao / p 2 o 5 ratio . in addition , many bioactive silica glasses are based upon the formula called “ 45s5 ”, signifying 45 wt % sio 2 and a 5 : 1 molar ratio of ca to p . glasses with substantially lower molar ratios of ca to p ( in the form of cao and p 2 o 5 ) do not bond to bone . however , substitutions in the 45s5 formula of 5 to 15 wt % b 2 o 3 for sio 2 or 12 . 5 wt % caf 2 for cao or ceraming the various bioactive glass compositions to form glass - ceramics have no measurable effect on the ability of the material to form a bone bond . the addition of even small amounts of al 2 o 3 , ta 2 o 5 , tio 2 . sb 2 o 3 , or zro 2 may inhibit the bonding to bone or tissue and , hence , generally are unsuitable as bone substitutes . illustrative bioactive glasses that are suitable as bone substitutes include : bioglass ®, novabone ™, and novabone c / m ™, all of which are products of us biomaterials ( alachua , fla .). similar to the bioactive glasses are the bioactive ceramics . it now is generally accepted that calcium - based ceramics may be used as bone substitutes . both calcium sulphate - and calcium phospate - based ceramics have been employed in this regard . to date , the calcium phosphate based - ceramics , such as hydroxyapatite - and hydroxylapatite - based ceramics , that most closely resemble the inorganic constituent of bone are the most effective bone substitutes . exemplary bioactive ceramics that can be used as bone substitutes are ceravital ®, pro osteon ™ coralline hydroxyapatite ceramic , a product of interpore international company , ( irvine , calif . ), crystalline hydroxyapatite ceramic , which is available commercially from zimmer scientific ( warsaw , ind . ), and a / w glass ceramic . the collagen that represents a second structural component of the invention is preferably type i collagen . type 1 collagen is commercially available from several sources , including sigma chemical ( st . louis , mo .) and worthington biochemical corporation ( lakewood , n . j .). in lieu of collagen per se , one also can employ a mammalian derived gelatin - or a collagen - containing bioabsorbable sponge such as gelfoam ™, a product of upjohn ( kalamazoo , mich . ), and surgifoam ™, a product of ethicon ( somerville , n . j .). these products are commercially available and can be added directly to the mixture of bone marrow cells and pulverized bone or bone substitute , in the context of creating an engineered bone marrow of the present invention . in order to prepare the engineered bone marrow of the present invention , bone marrow cells are combined with enough pulverized bone or bone substitute to provide a gelatinous composition . in one aspect of the invention , about 1 × 10 6 to about 5 × 10 8 , about 5 × 10 6 to about 1 × 10 8 , or about 1 × 10 7 to about 6 × 10 7 bone marrow cells are combined with about 50 mg to about 500 mg of pulverized bone or bone substitute . in another aspect of the invention , about 10 × 10 6 to about 20 × 10 6 bone marrow cells are combined with about 50 mg to about 200 mg of pulverized bone or bone substitute . next , sterile , collagen is added to the mixture of bone marrow cells and pulverized bone or bone - substitute . collagen obtained commercially usually is acidified to prevent collagen fibers from crosslinking and forming a matrix . for the present invention , in contrast , the formation of a collagen matrix is desired . accordingly , collagen that is added to a mixture of bone marrow cells and pulverized bone , according to the invention , preferably takes the form of a solution of neutralized collagen matrix , especially neutralized type i collagen matrix . methods for producing neutralized type 1 collagen matrix solution are detailed , for example , by eschenhagen et al ., faseb j . 11 : 683 - 94 ( 1997 ), and fink et al ., loc . cit . 14 : 669 - 79 ( 2000 ). the collagen concentration of the neutralized type 1 collagen matrix solution is about 1 mg / ml to about 2 mg / ml , preferably between about 1 . 3 mg / ml to about 1 . 5 mg / ml . in one embodiment , about 0 . 1 mg to about 1 . 5 mg of neutralized type 1 collagen matrix is added to the mixture of bone marrow cells and pulverized bone or bone substitute . in general , this requires an addition of about 100 μl to about 750 μl or preferably about 300 μl to about 500 μl of neutralized type i collagen matrix solution to the mixture of bone marrow cells and pulverized bone or bone substitute . in another embodiment , about 0 . 3 to about 1 . 0 mg of neutralized type 1 collagen matrix is added to the bone marrow composition . the weight of the bone marrow cells can be determined by centrifuging the cells and removing any liquid . in general , 11 − 13 × 10 6 bone marrow cell weigh about 53 . 5 to 65 . 8 mg , and an engineered bone marrow prepared using the above method has a weight ratio of about 8 - 98 % bone marrow cells , about 1 - 91 % pulverized bone or bone substitute , and about 0 . 004 - 1 . 0 % of collagen or collagen substitute . in a preferred embodiment , the engineered bone marrow has a weight ratio of about 32 % to about 91 % bone marrow cells , about 9 % to about 67 % pulverized bone or bone substitute , and about 0 . 01 % to 1 % of collagen or collagen substitute . in a more preferred embodiment , the engineered bone marrow has a weight ratio between about 48 to about 84 % bone marrow cells , about 16 % to about 52 % pulverized bone or bone substitute , and about 0 . 03 to about 1 . 0 % of collagen or collagen substitute . the inventors also have demonstrated that the transplantation site determines the efficiency of engraftment of the engineered bone marrow . the transplantation procedure requires implantation into a vascular tissue site , although not all vascular tissue sites allow efficient engraftment . implantation of the inventive composition in a highly vascular intramuscular site , for instance , did not allow survival of the engineered bone marrow . by contrast , the inventors have found that implantation at a site in the portal system typically leads to the formation of a bone marrow type structure , in accordance with the present invention . the portal system is that portion of the circulatory system that drains into the liver . preferably , a composition of the invention is implanted in an intramesenteric portal site of the portal system , that is , in a portion of the portal system associated with the small or large intestine . to this end , an intramesenteric pocket is created by folding the leaves of the mesentery over one another in order to create space for engraftment . a key consideration to successful clinical application of the inventive technology is the construction of an adequate volume of bone marrow , in order to support sufficiently high numbers of donor hematopoietic stem cells . for this purpose , the small bowel mesentery , with its large surface area , offers an ideal site to overcome space limitations . in one aspect of the invention , the ratio of the volume of engineered bone marrow composition implanted to the volume of mesentery is about 0 . 1 to about 0 . 9 to 1 . in a preferred embodiment , the ratio of the volume of engineered bone marrow composition implanted to the volume of mesentery is about 0 . 1 to about 0 . 75 to 1 . in a more preferred embodiment the ratio of the volume of engineered bone marrow composition implanted to the volume of mesentery is about 0 . 1 to about 0 . 5 to 1 . the following examples are given to illustrate the present invention . it should be understood , however , that the invention is not to be limited to the specific conditions or details described in these examples . cell isolation : all animals were housed in the laboratory animal facility of the abramson pediatric research center at the children &# 39 ; s hospital of philadelphia . all experimental protocols were reviewed and approved by the institutional animal care and use committee at the children &# 39 ; s hospital of philadelphia , and followed guidelines set forth in the national institutes of health guide for the care and use of laboratory animals . eight to twelve week old c57 / bl6 female mice ( jackson laboratories , bar harbor me .) were used as the source of both bone and bone marrow . after sacrifice by cervical dislocation , the hind limbs were removed under sterile conditions and cleaned of all muscular and connective tissue . bone marrow from the femoral and tibial cavities was flushed out with dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % heat inactivated fetal calf serum , 2 mm l - glutamine , 100 u / ml penicillin , and 100 mg / ml streptomycin sulfate ( gibco brl , gaithersburg , md .). the bone marrow was separated into a single cell suspension by sequential passage through 21 , 24 , and 26 gauge needles . the remaining long bones then are crushed mechanically to a fine suspension , using sterile scissors , and further degraded into a fine powder by sequential passage through a 16 - and then an 18 - gauge needle such as those sold by becton dickinson and company ( franklin lakes , n . j .). both the bone and marrow components were combined with the delivery vehicle in vitro prior to in vivo implantation . preparation of delivery vehicles : neutralized collagen extracellular matrix was prepared by combining 600 μl of type i rat tail collagen ( 3 . 69 mg / ml ) ( collaborative biomedical , bedford , mass .) with 100 μl of sterile 0 . 1n naoh , 350 μl of 3 × dulbecco &# 39 ; s modified eagle &# 39 ; s medium and 525 μl of 1 × dulbecco &# 39 ; s modified eagle &# 39 ; s medium with 30 % fetal calf serum . diluted matrigel ® basement membrane matrix , growth factor - reduced and phenol red - free ), a product of collaborative biomedical ( bedford , mass . ), was prepared by combining 400 μl of matrigel ® with 200 μl of 3 × dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 30 % fetal calf serum . both mixtures were kept on ice until implantation . a 1 mm thick sheet of 95 % porous , non - woven polyglycolic acid mesh ( pga ) ( davis & amp ; geck ) was cut into pieces approximately 0 . 25 cm 2 and three free edges of two adjacent pieces were sewn together in order to create a bilayer pocket . the mesh was sterilized by a four - hour submersion in 100 % isopropyl alcohol and overnight exposure to uv radiation in a tissue culture hood . the construct was washed several times in sterile dulbecco &# 39 ; s modified eagle &# 39 ; s medium prior to implantation . implantation procedure : the cell suspension obtained from the mechanical degradation of one to two hind limbs was combined with 500 μl of a hydrogel delivery vehicle consisting of either neutralized collagen matrix or the diluted matrigel ® basement matrix in a tuberculin syringe . by injecting an identical volume of the cell suspension between the two leaves of a polyglycolic acid construct ( pga ) and oversewing the remaining open edge a surgically implantable biocompatible construct was manufactured for implantation . due to the gelatinous nature of the crushed bone / bone marrow suspension , minimal cell loss occurred through the interstices of the pga mesh prior to transplantation . all constructs were kept on ice until implantation . recipient animals consisted of syngeneic c57 / bl6 mice ranging between 12 and 30 weeks of age . prior to both injection and implantation , the animals were anesthetized with metofane ™, a product of mallinckrodt veterinary inc . ( mundelein , ill . ), and the surgical site was prepped with betadine . only one site per animal was used for cell transplantation . subcutaneous injection of the collagen based construct into the left flank was done using a tuberculin syringe capped with a 16 gauge needle while the polyglycolic acid mesh based construct was surgically implanted into a subcutaneous pocket created by blunt dissection in the same location . intramuscular injection was carried out in a similar manner after exposure of the rectus abdominis muscle through a midline skin incision . implantation of the pga construct within the rectus abdominis musculature was accomplished by creating a pocket between the muscle layers . the area of injection or construct implantation was marked by 7 - 0 polypropylene sutures . for intramesenteric implantation a midline celiotomy was performed and the small bowel was eviscerated . after exposure of the mesentery down to its root , folding two leaves of the mesentery upon one another and securing them together with a 7 - 0 polypropylene suture created an intramesenteric pocket . in separate sets of experiments type i collagen and matrigel ® based constructs were injected into this pocket and allowed to harden prior to abdominal closure . the pga based construct was surgically implanted into this location using a 7 - 0 polypropylene suture . animals were allowed to recover with free access to food and water prior to analysis . morphologic analysis : animals in each experimental group were sacrificed at an early ( 3 to 6 weeks ) or a delayed time point ( 6 - 12 weeks ) after implantation . results are based on two to four animals analyzed per experimental group . after sacrifice by cervical dislocation , the area of cellular implantation was excised , fixed in 10 % buffered formalin , decalcified in cal - ex ( fisher scientific , pittsburgh , pa .) and embedded in paraffin . five - micron thick sections from representative areas were prepared and stained with hematoxylin and eosin . the slides were reviewed by light microscopy for cellular engraftment , the presence of bone marrow , and overall inflammatory response . immunohistochemistry : to further define the hematopoietic elements in the tissue engineered bone marrow immunohistochemical staining for hematopoietic stem cells was performed . cd 34 is a glycoprotein whose surface expression has previously been used to identify hematopoietic stem cells within bone marrow . after paraffin removal from five - micron thick sections of the tissue - engineered bone marrow , the slides were rehydrated for processing . after high temperature antigen retrieval , using antigen unmasking solution ( product of vector laboratories , burlingame , calif .) and appropriate blocking steps , the sections were incubated with rat anti mouse cd34 ( clone ram34 ), a product of pharmingen ( san diego , calif .) in 4 ° c . at a 1 : 10 dilution . after overnight incubation unbound antibody was washed off and the sections were incubated with biotinylated rabbit anti - rat antibody . immunoperoxidase development was accomplished after signal amplification by avidin : biotinylated enzyme complex kit , marketed by vector laboratories . positive reactivity was classified as dark brown staining with appropriate normal bone marrow sections serving as a positive control . transmission electron microscopy : after processing , fixation and decalcification of bone marrow constructs , 50 μm sections were cut from the paraffin sections and rehydrated . the constructs were then post - fixed in 2 % osmium with 0 . 1m sodium cacodylate followed by enblock stain in 2 % aqueous uranyl acetate . the tissue then was dehydrated in graded alcohol , clarified in propylene oxide and , after infiltrating with epon , embedded in the same resin . resin was heat cured at 70 ° c . for 48 hours and 70 nm thin sections were cut using a leica ultracut s microtome and diatome diamond knife . sections were picked up on 200 mesh thin bar grids , stained with saturated alcoholic uranyl acetate , counterstained in bismuth subnitrate and observed in jeol jem 1010 . images were captured using hamamatsu ccd camera and amt hr - 12 systems . subcutaneous injection of the cell suspension in a type i collagen gel and surgical implantation of the pga based cellular construct into a subcutaneous pocket resulted in the formation of a palpable nodule that persisted for the duration of the experiment . gross and histologic evaluation of specimens from two time points revealed minimal cellular survival , a devitalized bony matrix with a dense inflammatory response , and no marrow formation ( fig1 ). injecting or surgically implanting identical cellular constructs within the highly vascular intramuscular pocket increased survival of osteocytes within lacunae of the cancellous bone but did not result in bone marrow formation ( fig2 ). small bowel mesentery seeded with marrow stroma in a type i collagen gel had a palpable nodule in all animals studies . gross examination upon sectioning revealed numerous islands of bloody , gelatinous material resembling bone marrow ( fig3 ). histologic evaluation confirmed the presence of bone marrow with distinct islands of hematopoiesis separated by blood vessels , sinusoids , and adventitial reticular cells . upon closer examination megakaryocytes were also discernable ( fig4 , and 6 ). transmission electron microscopy revealed the presence of early hematopoietic precursors surrounding endothelial sinusoids and reticular cells ( fig7 ) and immunohistochemical evaluation revealed identifiable cd 34 positive hematopoietic stem cells within the tissue ( fig8 ). parallel rows of rope like type i collagen fibers could also be seen interspersed within the constructs ( fig9 ). when pga mesh rather than type i collagen gel was used as the delivery vehicle , no marrow formation was evident despite survival and engraftment of bone ( fig1 ). to test the role of type i collagen in the formation of bone marrow , matrigel ®, consisting of type iv collagen ( 30 %) and laminin ( 61 %), was used as the delivery vehicle to the mesenteric site in a portion of animals . no marrow formation was visible under these conditions .
an engineered bone marrow composition , preferably comprising bone marrow cells , pulverized bone , and type 1 collagen , can be transplanted into the portal system of a patient . the engineered bone marrow provides a microenvironment , for engraftment of hematopoietic stem cells , that increases levels of chimerism while decreasing or eliminating failure of engraftment .