Patent Publication Number: US-11649439-B2

Title: Biologic composition and method of manufacture

Description:
RELATED APPLICATIONS 
     This invention is a continuation of co-pending U.S. application Ser. No. 16/049,215 filed on Jul. 30, 2018 which is a continuation of U.S. application Ser. No. 15/591,513 filed on May 10, 2017 now U.S. Pat. No. 10,513,690 issued on Dec. 24, 2019 which is a division of U.S. application Ser. No. 14/683,221 filed on Apr. 10, 2015 entitled, “Biologic Composition And Method Of Manufacture” now U.S. Pat. No. 9,675,643 issued on Jun. 13, 2017 which claims benefit of provisional 62/129,351 filed on Mar. 6, 2015 and provisional 62/129,337 filed on Mar. 6, 2015. 
    
    
     TECHNICAL FIELD 
     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. 
     BACKGROUND OF THE INVENTION 
     In the area of tissue regeneration or repair, the use of stem cell therapy has been widely touted. 
     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. 
     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. 
     The present invention has yielded a biological composition that is safe and achieves high yields of viable stromal cells and does so in a method that allows the resultant mixture to be recovered in a non-expanded and non-differentiated way from bone marrow wherein the mixture unexpectedly exhibits increased CD105 and STR01 markers at time of use when compared to the quantity at the time of actual processing. This evidences a maintenance of viable cells in the dose, an increase in mesenchymal cells in the dose and a legacy or memory of the lineages from where the cells came which retain the ability to differentiate into new tissue forms other than bone. 
     These and other benefits of the present invention and the method of preparing it are described hereinafter. 
     SUMMARY OF THE INVENTION 
     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 is compatible with biologic function. 
     The mixture of mechanically selected material derived from bone marrow further can have non-expanded whole cells. The biological composition preferably has bone. The bone can be added to the mixture derived from bone marrow. The bone includes a mixture of cortical bone and cancellous bone. 
     The combination of non-whole cell components with a select number of the non-expanded cells sustains pluripotency in the cells. The select number of the non-expanded cells includes differentiated committed cells and non-differentiated and non-committed 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. 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. The gradient can have a chemical characteristic of spatially changing compositions of density or species of functional molecules. Also, the gradient can have an electrical characteristic of charge based or pH based or electron affinities that confer metastability in biologic potential. 
     The bone marrow mixture which is derived from a cadaver has separation-enhanced cell vitality including one or more of the following: separating the 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 can be 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 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. 
     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 cellular or non-cellular components or a combination thereof; washing the cellular or non-cellular components or a combination thereof to create the mixture; quantifying cell concentration not to exclude zero; suspending to a mixture in a cryoprotectant; freezing the mixture; and packaging a volume of bone within the mixture or separate. 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 being intermixed, can be implanted by packing, injection, scaffolding or any other suitable means into a patient. 
     Definitions 
     DNase—deoxyribonuclease is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. 
     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) 
     DPBS—Dulbecco&#39;s Phosphate Buffered Saline. 
     CBT-MIXER—Mixing blade for Cancellous Bone Tumbler Jar. 
     Cold Media—Media used during the preparation of vertebral bodies for initial processing. 
     Cryopreserved—Tissue frozen with the addition of, or in a solution containing, a cryoprotectant agent such as glycerol or dimethylsulfoxide. 
     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. 
     Normal Saline—0.9% Sodium Chloride Solution. 
     Packing Media—Media used during initial processing and storage of the processed vertebral bodies prior to bone decellularization. 
     PBS—Phosphate Buffered Saline. 
     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 
       The invention will be described by way of example and with reference to the accompanying drawings in which: 
         FIG.  1    shows a photograph of a cut vertebral body taken from a spine of a cadaver donor. 
         FIG.  2    shows a photograph of the vertebral body after being cut into cubic pieces and immersed in a packing media. 
         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. 
         FIG.  4    shows a photograph of the jar with a CBT-Mixer connected to a tumbler. 
         FIG.  5    is a photograph of an exemplary sieve device having sieves sized to separate the solid material. 
         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 cell interface layer. 
         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. 
         FIG.  8 A  is a chart showing the percent recovery at 6 months after cryofreezing the mixture of 1 ml at 1.1×10 6  cells and thawing. 
         FIG.  8 B  is a chart showing the viability at 6 months after cryofreezing and thawing. 
         FIG.  8 C  shows a chart at 6 months of MSC markers by percentage of cells. 
         FIGS.  9 A and  9 B  are photographs of cells thawed from a single sample and placed in media at 37 degrees C. overnight evidencing cell viability. 
         FIG.  10    is a representative photograph of the final packaging. 
         FIG.  11    is a photograph showing ground bone. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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   . 
     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   . 
     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. 
     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. 
     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   . 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                 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 500- 
                 Use the 500- 
                 Use the 500- 
                 Use the 500- 
               
               
                   
                 μm and the 
                 μm, 180-μm 
                 μm, 180-μm 
                 μm, 180-μm 
               
               
                   
                 bottom pan 
                 and bottom 
                 and bottom 
                 and bottom 
               
               
                   
                 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. 
               
               
                   
               
            
           
         
       
     
     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 . 
     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 commingling 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. 
     After this as shown in  FIG.  6   , the step of separating the cells  240  by a density centrifugation occurs. The mixture including whole cells  240  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 and the interface  230  including the desired components which can include whole cells  250  is then collected using a 5 ml pipette and transferred into new 50 ml conical tubes ensuring no tube has more than 10 ml. Then the volume is brought to 50 ml by adding DPBS and centrifuged at 400×g for 5 minutes at room temperature, preferably about 20 degrees C. and the supernatant is removed leaving a pellet. Each 50 ml tube is then filled up to 50 ml with DPBS to resuspend the pellet. Another centrifugation occurs and the supernatant is removed and the remaining pellet is resuspended using the process media with no antibiotics. The suspension is then used to resuspend all the pellets in remaining tubes. The suspension volume is brought to 50 ml by adding processing media with no antibiotic. Then the suspension can be strained using a 100 μm cell strainer if any visual clumping is seen. These steps effectively wash the cells  250 , if present, and the non-cellular components. A representative sample is then counted. The remaining, or a portion thereof, of the cellular or non-cellular components or a combination thereof is centrifuged and resuspended in the desired protectant after which it&#39;s placed in vials holding 1 ml. 
     In the preferred embodiment, this results in 1.1×10 6  cells per ml, but could cover any concentration from zero to less than 5.0×10 6  cells per ml depending on the desired concentration wanted per cc. 
     Once the cell count is established and each 1 ml suspension is established or quantified, the material is taken and suspended in a predetermined concentration of a polyampholyte cryoprotectant or any other suitable alternative protectant. When using the cryoprotectant, a freezing of the mixture at a predetermined control rate is required. Ideally, the application of a cryoprotectant coats each cell  250  and provides a protective coating to keep the cell viable during the freezing process. While the techniques for cryopreservation are well known, the present invention after being frozen has demonstrated remarkably unexpected results. 
     When thawed and a cell count is preformed after manufacture, the cell viability is 80 percent. Thawing is in a water bath warmed to 37 degrees C. for 2-3 minutes. After storage for 6 months, the cell viability is 91.0+/−3.8%. The percent recovery from freeze at 6 months thaw is 82.8+/−7.2%. The inventors have noted that the recovery count is lower than the viability to the lysis of undesirable GlycoA+ cells during freeze, a well-known occurrence. The unlysed desirable cells were viable at 91.0%. The inventors would also like to note that while thawed cells are generally suspended in FDS-supplemented media and spun, to better simulate how the product is actually used the cell recovery at six months was thawed and suspended in 3 ml of saline yielding a 4 ml suspension and that was not spun, but measured directly to simulate a real use injection. This allowed the cryopreservative to more effectively demonstrate that actual count of viable cells a patient would expect to receive and provides one explanation for this remarkable viability result. As shown in  FIGS.  9 A and  9 B , the cells  250  are shown under magnification. In the cells at 6 months thaw the percent of positive cells for MSC markers, specifically CD105 and STRO1+ are 52 percent and 74 percent respectively. These indicate the majority of cells are non-differentiated and directionally favorable for new bone formation. 
     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. 
     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. 
     In one embodiment, the composition includes the whole cells in the mixture. In that embodiment, it is possible to provide bone with the mixture either in the mixture or separately to be combined at the time of use. 
     In one embodiment, the bone is ground to a size of 100 to 300 μm, see  FIG.  11   . Other ranges of bone sizes and mixtures can be employed depending on the application which, in this example, was bone regeneration. 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 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. 
     The bone mixture may be cortical or cancellous or any combination of both. The bone can be mineralized or demineralized or any combination of both. The bone may be in any form or size or shape suitable for bone repair. Lower volumes and cell counts 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. 
     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.