the term “ tissue ” is used in the general sense herein to mean any transplantable or implantable tissue , the survivability of which is improved by the methods described herein upon implantation . in particular , the overall durability and longevity of the implant are improved , and host - immune system mediated responses , are substantially eliminated . the terms “ transplant ” and “ implant ” are used interchangeably to refer to tissue , material or cells ( xenogeneic or allogeneic ) which may be introduced into the body of a patient . the terms “ autologous ” and “ autograft ” refer to tissue or cells which originate with or are derived from the recipient , whereas the terms “ allogeneic ” and “ allograft ” refer to cells or tissue which originate with or are derived from a donor of the same species as the recipient . the terms “ xenogeneic ” and “ xenograft ” refer to cells or tissue which originate with or are derived from a species other than that of the recipient . the term “ growth factor ” means a naturally occurring or synthetic compound capable of stimulating cellular proliferation and / or cellular differentiation . growth factors are important for regulating a variety of cellular processes . the term “ elisa ” or “ enzyme - linked immunosorbent assay ” means a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample . the elisa has been used as a diagnostic tool in medicine and plant pathology , as well as a quality control check in various industries . in simple terms , in elisa an unknown amount of antigen is affixed to a surface , and then a specific antibody is washed over the surface so that it can bind to the antigen . this antibody is linked to an enzyme , and in the final step a substance is added that the enzyme can convert to some detectable signal . thus in the case of fluorescence elisa , when light is shone upon the sample , any antigen / antibody complexes will fluoresce so that the amount of antigen in the sample can be measured . the present invention is directed towards an osteochondral repair construct constructed of cancellous bone taken from allogenic or xenogenic bone sources . the construct is preferably derived from dense allograft cancellous bone that may originate from proximal or distal femur , proximal or distal tibia , proximal humerus , talus , calcaneus , patella , or ilium . cancellous tissue is first processed into blocks and then milled into the desired shapes such as a cylinder for this present invention . a preferred embodiment of the assembled construct 10 is illustrated in fig3 . a cancellous bone cylinder is milled using a lathe to form an unbalanced barbell - shaped , primary base member 12 , as illustrated in fig3 and 4 . the primary base member 12 includes a top section 14 , a cylindrical stem section 16 and a cylindrical base section 18 . the top section 14 is milled to have a thickness similar to the thickness of human articular cartilage ( e . g ., 1 . 5 - 3 . 5 mm ) and the and the diameter of the implant may vary between 5 - 25 mm . the stem section 16 has a diameter approximately half of the diameter of the entire assembled construct 10 . the base section 18 has a thickness or length which is preferably larger than the thickness or length of the top section 14 with a ratio preferably ranging from of at least about 1 . 5 to 1 to about 6 : 1 . during tissue processing , the top section 14 is substantially demineralized by immersing it in dilute acid while the base section 18 remains mineralized . reference is now made to fig3 and 5 , which illustrate a ring - shaped secondary member 20 having an aperture 22 with a diameter equal to or slightly greater than the diameter of the stem 16 and an outer diameter which is the same as the diameter of the top section 14 and base section 18 . however , if desired , the aperture 22 can be 10 % to 40 % larger than the diameter of the stem 16 . the top surface 24 and bottom surface 26 of the ring - shaped , or ring , member 20 are preferably planar and after assembly the bottom surface 26 is adjacent the top surface 19 of the base section 18 and the top surface 24 is adjacent the bottom surface 15 of the top section 14 . while the ring member 20 is preferably constructed of mineralized allograft cancellous bone , it can be constructed of allograft cortical bone or xenograft bone as long as the same have been decellularized . alternately , the ring member 20 may be constructed of ceramics or biocompatible polymers . the top section 14 is substantially demineralized in dilute acid up to a predetermined level ( as indicated by broken - line representation l 1 in fig7 ) until the bone contains less than 0 . 5 % wt / wt residual calcium . subsequently , the resultant tissue form is predominantly type i collagen , which is sponge - like in nature with an elastic quality . following decalcification , the tissue is further cleaned , brought to a physiological ph level of about 7 and may also be treated so that the cancellous tissue is non - osteoinductive . this inactivation of inherent osteoinductivity may be accomplished via chemical or thermal treatment or by high energy irradiation . the cancellous top section 14 is preferably treated with an oxidizing agent such as hydrogen peroxide in order to render it non - osteoinductive . following demineralization the top section 14 is spongy and deformable allowing it to be squeezed through the center aperture 22 of the ring member 20 . after the implant has been assembled , morselized cartilage particles combined with a carrier or growth factor may be added to the top section 14 . if desired , the open cancellous structure of the top section 14 may be loaded with a cartilage paste or gel as noted below and / or one or more additives namely recombinant or native or variant growth factors ( fgf - 2 , fgf - 5 , fgf - 7 , fgf - 9 , fgf - 11 , fgf - 21 , igf - 1 , tgf - β1 , bmp - 2 , bmp - 4 , bmp - 7 , pdgf , vegf ), human allogenic or autologous chondrocytes , human allogenic cells , human allogenic or autologous bone marrow cells , human allogenic or autologous stem cells , demineralized bone matrix , insulin , insulin - like growth factor - 1 , interleukin - 1 receptor antagonist , hepatocyte growth factor , platelet - derived growth factor , indian hedgehog , parathyroid hormone - related peptide , viral vectors for growth factor or dna delivery , nanoparticles , or platelet - rich plasma . this design enables the fabrication of an implant that possesses a relatively uniform substantially demineralized top section that is distinct from the mineralized base section . the demineralized portion of the construct can be provided with a matrix of cartilage putty or gel consisting of minced or milled allograft cartilage which has been lyophilized so that its water content ranges from about 0 . 1 % to about 8 . 0 % ranging from about 25 % to about 50 % by weight , mixed with a carrier of sodium hyaluronate solution ( ha ) ( molecular weight ranging from about 7 . 0 × 10 5 to about 1 . 2 × 10 6 ) or any other bioabsorbable carrier such as hyaluronic acid and its derivatives , gelatin , collagen , chitosan , alginate , buffered pbs , dextran cmc , or other polymers , the carrier ranging from about 75 % to about 25 % by weight . in one embodiment , the cartilage is minced or milled to a size less than or equal to about 212 μm . in another embodiment , the cartilage is minced or milled to a size of from about 5 μm to about 212 μm . in another embodiment , the cartilage is minced or milled to a size of from about 6 μm to about 10 μm . in another embodiment , the cartilage can be minced or milled to a size of less than or equal to about 5 μm . the small size of the minced or milled particulate cartilage can facilitate increased exposure or release of various growth factors due to the increased aggregate surface area of the particulate cartilage used . the cartilage particles can contain endogenous growth factors . these endogenous growth factors can be extracted from the cartilage particles by the method outlined in example 1 and detected by the method outlined in example 2 . exogenous growth factors can also be combined with the cartilage particulate . in one embodiment , cartilage is recovered from deceased human donors , and the tissue may be treated with any known method or methods for chemically cleaning or treating a soft tissue . the cartilage is then lyophilized , milled , then sieved to yield particle sizes of , on average , less than or equal to 212 microns . the cartilage particles are mixed with a growth factor in an aqueous vehicle , then the particles can either be lyophilized and stored dry at room temperature or frozen , or used immediately . for example , particles containing chondrogenic growth factor can be added to any portion of the allograft construct , and particles containing osteogenic growth factor can be added to any portion of the allograft construct save the demineralized cancellous cap . the mixture containing the cartilage particles and growth factor can be lyophilized for storage . the growth factor can be any one of a variety of growth factors known to promote wound healing , cartilage andior bone development ( e . g . bmp &# 39 ; s partictularly bmp - 2 , fgf &# 39 ; s particularly fgf - 2 and - 9 and / or variants of fgf - 2 , igf , vegf , pdgf , etc .). the vehicle used to solubilize the growth factor and adsorb it into the cartilage particles can be saline , water , pbs , ringers , etc . in one embodiment , the resulting enhanced cartilage particles can contain levels of growth factors that are greater than that found in intact cartilage . in another embodiment , the cartilage particle mixture can be infused into all or part of the construct . if desired , the cartilage particle mixture can be infused primarily into the demineralized end of the primary member of the construct . it is further envisioned that cells which have been collected from the patient or grown outside the patient can be inserted into the entire construct or into the cancellous demineralized top section 14 matrix before , during or after deposit of the construct 10 into the defect area . such cells include , for example , allogenic or autologous bone marrow cells , stem cells and chondrocyte cells . the cellular density of the cells preferably ranges from 1 . 0 × 10 8 to 5 . 0 × 10 8 or from about 100 million to about 500 million cells per cc of putty or gel mixture . the construct 10 is placed in an osteochondral defect area bore which has been cut in the lesion area of a patient with the upper surface 17 of the top section 14 being slightly proud ( i . e ., above ), slightly below , or substantially flush with the surface of the original cartilage surrounding the defect area remaining at the site being treated . the construct 10 has a length which can be the same as the depth of the defect or more or less than the depth of the bore . if the construct 10 is the same as the depth of the bore , the base of the implant is supported by the bottom surface of the bore and the top surface 17 is substantially level with the articular cartilage . if the construct 10 is of a lesser length , the base of the construct is not supported but support is provided by the wall of the defect area bore or respective cut out area as the plug is interference fit within the bore or cut out area with the cap being slightly proud , slightly below , or flush with the surrounding articular cartilage depending on the surgeon &# 39 ; s preference . with such load bearing support , the graft surface is not damaged by weight or bearing loads which can cause micromotion interfering with the graft interface producing fibrous tissue interfaces and subchondral cysts . in operation , the lesion or defect is removed by cutting a blind bore removing a lesion in the implant area . the construct 10 is then placed in the bore or cut away area in an interface fit with the surrounding walls . if the construct is moveable within the bore , suitable organic glue material can be used to keep the implant fixed in place in the implant area . suitable organic glue material can also be used to keep the additives in the construct within the construct following implantation into the defect site . suitable organic glue material can be found commercially , such as for example : tisseel 7 or tissucol 7 ( fibrin based adhesive ; immuno ag , austria ), adhesive protein ( sigma chemical , usa ), dow corning medical adhesive b ( dow corning , usa ), fibrinogen thrombin , elastin , collagen , casein , albumin , keratin and the like . cartilage is recovered from deceased human donors , and the tissue may be treated with any known method or methods for chemically cleaning or treating a soft tissue . the cartilage is then lyophilized , freeze - milled , then sieved to yield particle sizes of , on average , less than or equal to 212 microns ,. the cartilage particles are - again lyophilized prior to storage or extraction . the particles are extracted in guanidine hcl by incubating at 4 ° c . on an orbital shaker at 60 rpm for 24 hr , followed by dialysis ( 8 k mwco membrane dialysis tube ) in 0 . 05m tris hcl for 15 hrs at 4 ° c . the dialysis solution was then replaced and the dialysis continued for another 8 hrs at 4 ° c . the post - dialysis extracts were stored at − 70 ° c . until elisa analysis . 0 . 25 g of cartilage particles were weighed out for each donor . the cartilage particles were transferred to tubes containing 5 ml of extraction solution ( 4m guanidine hcl in trishcl ). the cartilage particles were incubated at 4 ° c . on the orbital shaker at 60 rpm for 24 hr , followed by dialysis ( 8 k mwco membrane dialysis tube ) in 0 . 05m trishcl for 15 hrs at 4 ° c . the dialysis solution was then replaced and the dialysis continued for another 8 hrs at 4 ° c . the post - dialysis extracts were stored at − 70 ° c . until elisa run . notably , the above protocol can also be utilized in order to determine the total growth factor concentration ( e . g ., exogenous plus endogenous ) present in a device of the instant invention . fig8 illustrates the relative concentration of endogenous tgf - β1 found in cartilage particles of the present invention as derived from various donors and manufactured in accordance with example 1 . fig9 illustrates the relative concentration of endogenous fgf - 2 found in cartilage particles of the present invention as derived from various donors and manufactured in accordance with example 1 . fig1 illustrates the relative concentration of endogenous bmp - 2 found in cartilage particles of the present invention as derived from various donors and manufactured in accordance with example 1 . the results shown in fig8 indicate that processed cartilage particles prepared in accordance with the method of example 1 retain a concentration of endogenous tgf - β1 . the results shown in fig9 indicate that processed cartilage particles prepared in accordance with the method of example 1 also retain concentrations of endogenous fgf - 2 . the results shown in fig1 indicate that processed cartilage particles prepared in accordance with the method of example 1 also retain concentrations of endogenous bmp - 2 . the principles , preferred embodiments and modes of operation of the present invention have been described in the foregoing specification . however , the invention should not be construed as limited to the particular embodiments which have been described above . instead , the embodiments described here should be regarded as illustrative rather than restrictive . variations and changes may be made by others without departing from the scope of the present invention as defined by the following claims .
the invention is directed toward an osteochondral repair assembly comprising a shaped allograft construct comprising an unbalanced barbell - shaped cylindrical cancellous bone primary member formed with a mineralized cylindrical base section having a smaller diameter cylindrical stem leading to a second cylindrical section which is demineralized . a mineralized ring - shaped support member is forced over the compressed demineralized second demineralized the aperture of the ring - shaped member to fit around the stem with one ring surface being adjacent the bottom surface to the second cylindrical section and the opposite ring surface being adjacent the upper surface of the mineralized cylindrical base section .