Abstract:
A biological composition has a mixture of mechanically selected allogeneic biologic material derived from bone marrow. The mixture has non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. The mixture including non-whole cell fractions including one or more of exosomes, transcriptosomes, proteasomes, membrane rafts, lipid rafts. The mixture is compatible with biologic function.

Description:
RELATED APPLICATIONS 
       [0001]    This application is a division of co-pending U.S. application Ser. No. 14/682,523 filed on Apr. 9, 2015 entitled “Acellular Biologic Composition And Method Of Manufacture”. 
     
    
     TECHNICAL FIELD 
       [0002]    This invention is a tissue regenerative biological composition. More specifically, a composition at least in part formed from bone marrow and a method of manufacture and use of said composition with an acellular mixture. 
       BACKGROUND OF THE INVENTION 
       [0003]    In the area of tissue regeneration or repair, the use of stem cell therapy has been widely touted. 
         [0004]    Often, these inventions describe isolating the stem cells, purifying and culturally expanding mesenchymal stem cells. In U.S. Pat. No. 5,837,539, entitled “Monoclonal Antibodies For Human Mesenchymal Stem Cells”, Arnold Caplan et al. reported that the cells are preferably culturally expanded, but suggest it is possible to use the stem cells without culture expansion. Caplan also describes a way to isolate stem cells. 
         [0005]    A major technological hurdle to producing a safe allogeneic composition with viable cells has been the need to approach a fraction of risk approaching zero by removing all antigenic properties that lead to inflammation factors in a separation to yield only a certain stromal cell type. This has proven both difficult and degrading the quantity of viable cells that can be effectively harvested. 
         [0006]    The present invention has yielded a biological composition that is safe and achieves and does so in a method that allows the resultant mixture to be recovered from bone marrow wherein the mixture unexpectedly exhibits evidence of viability independent of mesenchymal cells in the dose and sustains a legacy or memory of the lineages from where the acellular biological composition came which retain the ability to support the emergence of new tissue forms including bone and other tissues. 
         [0007]    These and other benefits of the present invention and the method of preparing it are described hereinafter. 
       SUMMARY OF THE INVENTION 
       [0008]    A biological composition has a mixture of mechanically selected allogeneic biologic material derived from bone marrow. The mixture has non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. The mixture including non-whole cell fractions including one or more of exosomes, transcriptosomes, proteasomes, membrane rafts, lipid rafts. The mixture is compatible with biologic function. 
         [0009]    The mixture of mechanically selected material derived from bone marrow. The biological composition preferably has bone particles. The bone particles can be added to the mixture derived from bone marrow. The bone particles include a mixture of cortical bone particles and cancellous bone particles. 
         [0010]    The combination of non-whole cell components with a select number of non-whole cell fractions sustains pluripotency in the cells. In a preferred embodiment, the biological composition is predisposed to demonstrate or support elaboration of active volume or spatial geometry consistent in morphology with that of endogenous bone. The biological composition extends regenerative resonance that compliments or mimics tissue complexity. The mixture is treated in a protectant or cryoprotectant prior to preservation or cryopreservation or freeze drying. The composition can be maintained at ambient temperature prior to freeze drying. The protectant or cryoprotectant creates a physical or electrical or chemical gradient or combination thereof for tissue regeneration. The gradient can have a physical characteristic of modulus or topography, such as charge density, field shape or cryo or chemo toxic tendencies. The gradient can have a chemical characteristic of spatially changing compositions of density or species of functional molecules, wherein the molecules can offer a fixed catalytic function as a co-factor. Also, the gradient can have an electrical characteristic of charge based or pH based or electron affinities that confer metastability in biologic potential. 
         [0011]    The bone marrow mixture which is derived from a cadaver has separation-enhanced non-whole cell fractions vitality including one or more of the following: separating the fractions from cells heightens their vitality, reversing “arrest” of donors, responsive molecular coupling, matrix quest in neutralizing inflammation or satience by balancing stimulus for repair. The protectant or cryoprotectant is a polyampholyte. The regenerative resonance occurs in the presence or absence of a refractory response. When using a cryoprotectant, the cryopreservation occurs at a temperature that is sub-freezing wherein the cryopreservation temperature is from 0 degrees C. to −200 degrees C. The protection may also be achieved by non-cryogenic means. 
         [0012]    The biological composition&#39;s non-whole cellular component also can include organelle fragments and the active and inactive components of biological activity which can also include extants of the human metabolome. 
         [0013]    A method of making a biological composition of the present invention has the steps of: collecting, recovering and processing bone marrow from a cadaver donor; mechanically separating the cellular or non-cellular components or a combination thereof of bone marrow from cadaverous bone; concentrating by centrifugation and filtering; separation by density gradient centrifugation; collecting non-cellular fractions or non-cellular components or a combination thereof of predetermined density; washing the non-whole cellular fractions or non-cellular components or a combination thereof to create the mixture; quantifying concentrations of non-cellular fractions components at a non-zero entity; suspending to a predetermined concentration in a polyampholyte cryoprotectant; freezing the mixture at a predetermined controlled rate; and packaging a bone blend having particles in the size range of 100 to 300 μm of demineralized cortical bone, mineralized cortical bone and mineralized cancellous bone either within the mixture or separate. These particle size ranges can vary higher or lower depending on the application. At the time of use, the mixture is thawed by immersion in a warm water bath for 2-3 minutes at 37 degrees C. It is diluted in saline without spinning; and then the diluted mixture, with or without the bone blend being intermixed, can be implanted by packing, injection, scaffolding or any other suitable means into a patient. 
       Definitions 
       [0014]    DNase—deoxyribonuclease is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. 
         [0015]    DMEM, DMEM/LG—Dulbecco&#39;s Modified Eagle Medium, low glucose. Sterile, with: Low Glucose (1 g/L), Sodium Pyruvate; without: L-glutamine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 
         [0016]    DPBS—Dulbecco&#39;s Phosphate Buffered Saline. 
         [0017]    CBT-MIXER—Mixing blade for Cancellous Bone Tumbler Jar. 
         [0018]    Cold Media—Media used during the preparation of vertebral bodies for initial processing. 
         [0019]    Cryopreserved—Tissue frozen with the addition of, or in a solution containing, a cryoprotectant agent such as glycerol, or dimethylsulfoxide, or carboxylated poly-1-lysine. 
         [0020]    Freeze Dried/Lyophilized—Tissue dehydrated for storage by conversion of the water content of frozen tissue to a gaseous state under vacuum that extracts moisture. 
         [0021]    Normal Saline—0.9% Sodium Chloride Solution. 
         [0022]    Packing Media—Media used during initial processing and storage of the processed vertebral bodies prior to bone decellularization. 
         [0023]    PBS—Phosphate Buffered Saline. 
         [0024]    Processing Media—Media used during bone decellularization that may contain DMEM/Low Glucose no phenol red, Human Serum Albumin, Heparin, Gentamicin and DNAse. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0025]    The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The invention will be described by way of example and with reference to the accompanying drawings in which: 
           [0026]      FIG. 1  shows a photograph of a cut vertebral body taken from a spine of a cadaver donor. 
           [0027]      FIG. 2  shows a photograph of the vertebral body after being cut into cubic pieces and immersed in a packing media. 
           [0028]      FIG. 3  shows a photograph of the bulk bone material after being ground and immersed in packing media and placed in a jar for later tumbling. 
           [0029]      FIG. 4  shows a photograph of the jar with a CBT-Mixer connected to a tumbler. 
           [0030]      FIG. 5  is a photograph of an exemplary sieve device having sieves sized to separate the solid material. 
           [0031]      FIG. 6  shows a photograph of two 50 ml vials, the one on the left being prior to centrifuging with the Ficoll that is commercially available at the bottom and the material above it. The 50 ml vial on the right is after centrifuging showing the non-whole cell fraction interface layer. 
           [0032]      FIG. 7  is a photograph showing the four tumbling steps  1 - 4  by exemplary collection and Ficoll separation of the decanted fluids, the fluid in tumble  1  being completely discarded to remove unwanted debris. 
           [0033]      FIG. 8  shows a photograph of two 50 ml vials, the one on the left being prior to centrifuging with a sucrose gradient that is commercially available at the bottom and the material above it. The 50 ml vial on the right is after centrifuging showing the non-whole cell fraction above the interface layer. 
           [0034]      FIG. 9  is a representative photograph of the final packaging. 
           [0035]      FIG. 10  is a photograph showing the ground bone. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0036]    With reference to the present invention which is a tissue regenerative biological composition  100  made from bone marrow  200 , it is believed best understood by the methods used to process and recover the biological composition, as illustrated in the  FIGS. 1-6 . 
         [0037]    The first steps are to collect, recover and process bone marrow  200  from a cadaver donor. To do this, the spine is removed aseptically from the cadaver and the resultant spine segment is covered by cold media. The cold media has 0.5 ml of Heparin; 10,000 units/ml per 500 ml of DMEM. DMEM is a sterile solution with low glucose (1 g/L), Sodium Pyruvate; without L-glutamine, or HEPES. This cold media is used for packaging the spine segments for later processing. At this point the spine segment includes a plurality of vertebral bodies  202 . The clinical technician must remove as much soft tissue as possible and cut each vertebral body  202  with a saw. These vertebral bodies  202 , once cleaned, of all adherent soft tissue around the cortical surfaces will look as shown in  FIG. 1 . 
         [0038]    Once a cleaned vertebral body  202  is obtained, the next step involves cutting each vertebral body  202  into pieces, each piece  204  roughly 1 cm 3 . The cut pieces  204  being immersed in a packing media  400 . The exemplary packing media can be DMEM with 0.5 ml Heparin and 1.25 ml of DNAse added. 
         [0039]    Once all the vertebral bodies  202  have been cut, the pieces  204  are taken to the bone grinder. The bone is ground into 4-10 mm pieces using packing media  400  to help the pieces go through the grinder. The ground bone  206  (bulk cortical-cancellous crushed) and all of the packing media  400 , estimated volume of 500 ml are transferred into a jar  300  where 0.5-1.0 ml of Gentamicin is added to the jar  300  with ground bone  206  and packing media  400 . At this point, the crushed bone  206 , including cellular soft marrow  200 , is intermixed. 
         [0040]    The step of mechanically separating these cellular components of bone marrow  200  from the cadaverous bone is next performed. Transferring the bulk cortical-cancellous bone chips into a new jar with a CBT-Mixer in the jar. The bulk cortical-cancellous bone chips  206  will go through four cycles as summarized in the table below. Each cycle, after cycle  1 , contains three steps using a bone tumbler  500  and sieve set  600 . The sieve set  600  has screens  602  of various sizes, for example 500 μm and 180 μm, as shown in  FIG. 5 . 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                 Step  
                 Cycle 1  
                 Cycle 2  
                 Cycle 3  
                 Cycle 4 
               
               
                   
               
             
             
               
                 Bone  
                 30 minutes.  
                 30 minutes  
                 30 minutes  
                 30 minutes  
               
               
                 Tumbler  
                 Using  
                 Using  
                 Using  
                 Using  
               
               
                   
                 500 mL 
                 500 mL  
                 500 mL  
                 400 mL  
               
               
                   
                 Processing  
                 Processing  
                 Processing  
                 Processing  
               
               
                   
                 Media  
                 Media  
                 Media  
                 Media  
               
               
                 Sieve Set  
                 Use the  
                 Use the  
                 Use the  
                 Use the  
               
               
                   
                 500-μm  
                 500-μm,  
                 500-μm,  
                 500-μm,  
               
               
                   
                 and the  
                 180-μm  
                 180-μm  
                 180-μm  
               
               
                   
                 bottom  
                 and bottom  
                 and bottom  
                 and bottom  
               
               
                   
                 pan sieve.  
                 pan sieve.  
                 pan sieve.  
                 pan sieve.  
               
               
                   
                 Discard  
                 Collect  
                 Collect  
                 Collect  
               
               
                   
                 decanted  
                 decanted  
                 decanted  
                 decanted  
               
               
                   
                 fluid. 
                 fluid. 
                 fluid. 
                 fluid. 
               
               
                 Centrifuge  
                 N/A  
                 Use  
                 Use  
                 Use  
               
               
                   
                   
                 decanted  
                 decanted  
                 decanted  
               
               
                   
                   
                 fluid.  
                 fluid.  
                 fluid. 
               
               
                   
               
             
          
         
       
     
         [0041]    In cycle  1 , the decanted fluid  210  is discarded. To best understand this, an exemplary  FIG. 7  shows conical tubes with the decanted fluids after each cycle followed by Ficoll separation. Tumble  1  or Cycle  1  has most of the unwanted cells and debris as evidenced by its dark and red appearance whereas each subsequent cycle  2 ,  3  and  4  are progressively cleared. This  FIG. 7  is only to illustrate the effects of multiple tumbles  1 - 4  and the value in discarding the decanted liquid  210  after the first tumble  1 . 
         [0042]    After each subsequent sieving of the bulk bone material  206 , the decanted fluid  212 ,  214 ,  216  containing the mixture with whole cells is collected and put into a collection jar. When the next three cycles are complete and the decanted fluid is all placed in the collection jar comingling the fluids  212 ,  214  and  216  to form a decanted fluid  220 . Then the centrifugation of the combined decanted fluid  220  occurs by placing the fluid  220  in a number of 250 ml conical tubes using a 100 ml pipette. The centrifuge is programmed to 280×g for 10 minutes at room temperature, preferably about 20 degrees C. The fluid  220  is passed through a blood filter to further remove any bone or spicules or clumps from the suspended cells. This completes the step of centrifuging and filtering. At this point, the mixture including whole cells  240  has been separated from the soft marrow tissue  200  and the remaining cancellous and cortical bone is discarded. 
         [0043]    After this as shown in  FIG. 6 , the step of separating the cells  240  from the non-whole cellular components by a density centrifugation occurs. The whole cells  240  are in the interface and the non-whole cell components are in the supernatant above the interface. The mixture including is placed in 50 ml conical tubes 20 with Ficoll 800 and undergoes a Ficoll-Paque separation under centrifugation wherein a cell density gradient is established by spinning at 400×g for 30 minutes at room temperature, preferably about 20 degrees C. The mixture includes cellular or non-cellular components or a combination thereof. All fluid  211  above the interface is removed include the desired non-whole cell components which exclude the whole cells  240 ,  250 . 
         [0044]    Typically, non-whole cell fragments, or membrane components have a diameter of 40-100 nm and can be separated within a density of 1.13-1.19 g/mL in a sucrose solution, and can be sedimented by centrifugation at 100,000 g. In fact, these fragments, or cell fractions, or microvesicles, have been collectively referred to as exosomes. Ranging in size from 20-1000 nm in diameter, they have been similarly referred to as nanoparticles, microparticles, shedding microvesicles, apoptotic blebs, and human endogenous retroviral particles. There are few firm criteria distinguishing one type of microvesicle from the other. 
         [0045]    Following removal of the cell fraction, the supernatant is further filtered through 0.45 and 0.2 μm filters. Exosomes are further collected and separated within the suspension in multiple centrifugation steps with increasing centrifugal strength to sequentially pellet cells (300 g), microvesicles (10,000 g) and ultimately exosomes (100,000 g). Cells are deliberately removed to achieve the non-whole cell fragments and microvesicles. 
         [0046]    Subsequent separation using density gradient-based isolation, using sucrose or commercially available prep can be applied to obtain more pure exosome preparations. Recent reports encouraging the use of iodixanol-based gradients for improved separation of exosomes from viruses and small apoptotic bodies are considerations left open to be adopted or adapted in refinement. Differing from sucrose, iodixanol forms iso-osmotic solutions at all densities, thus better preserving the size of the vesicles in the gradient, and both technologies are available to best isolation technology. In addition to these traditional isolation techniques, easy-to-use precipitation solutions, such as ExoQuick™ and Total Exosome Isolation™ (TEI), that have been commercialized reduce the need for expensive equipment or technical know-how. Although their mode-of-action has not been disclosed or validated, these kits are commonly used. 
         [0047]    Once the mixture is completed, the method can include additional steps. This leads to the use of a bone blend  102  shown in  FIG. 10 , preferably from the same vertebral bone or at least bone from the same donor. 
         [0048]    When the mixture is prepared, it can have whole cells or even no whole cells, but will have the mechanically selected non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. 
         [0049]    In one embodiment, the composition includes the whole cells in the mixture. In that embodiment, it is possible to provide bone particles with the mixture either in the mixture or separately to be combined at the time of use. 
         [0050]    In one embodiment, the bone is ground to a particle size of 100-300 μm, see  FIG. 11 . The bone mixture has 1.5 cc of mineralized cancellous bone  104 , 1.5 cc of mineralized cortical bone  105  and 2.0 cc of demineralized cortical bone  106  yielding 30 percent, 30 percent and 40 percent respectively of the total 5 cc (5 gram) of bone material  102 . The ranges coincide with the 1 cc of mixture when resuspended in 3 cc of saline to provide a bone particle and mixture for implantation, which can be by packing, injection, scaffolding or any other suitable means, into a patient in a fracture healing procedure, by way of example. 
         [0051]    Other ranges of bone particle sized and mixture can be employed depending on the application which, in this example, was bone regeneration. Lower volumes and concentrations may be more suited for less intrusive bone repairs or more if larger if larger amounts of material are needed as in a hip defect or repair. 
         [0052]    Variations in the present invention are possible in light of the description of it provided herein. While certain representative embodiments and details have been shown for the purpose of illustrating the subject invention, it will be apparent to those skilled in this art that various changes and modifications can be made therein without departing from the scope of the subject invention. It is, therefore, to be understood that changes can be made in the particular embodiments described, which will be within the full intended scope of the invention as defined by the following appended claims.