Method and device for activating stem cells

Abstract

Invention embodiments described herein include methods and devices for stimulating mesenchymal stem cells in a stem cell source to differentiate into osteoblasts capable of forming bone. Devices and methods described include exposing a stem cell source, such as bone marrow aspirate, adipose tissue and/or purified allogenic stem cells, to an active agent, in a manner effective to form activated stem cells.

1 . A method for preparing a implant composition for promoting bone growth in a mammal, comprising (a) contacting stem cells with one or more active agents for 24 hours or less to prepare an activated stem cells, (b) separating the active agents from the activated stem cells to form an activated stem cell population that is substantially free of active agents, and (c) mixing the activated stem cell population that is substantially free of active agents with a bone graft substitute to thereby prepare an implant composition for promoting bone growth in a mammal, wherein at least one active agent promotes differentiation of stem cells into osteogenic cells or osteogenic precursor cells. 
     
     
         2 . The method of  claim 1 , wherein the stem cells are contacted with one or more active agents for 5 minutes to 1 hour. 
     
     
         3 . The method of  claim 1 , wherein the stem cells are contacted with one or more active agents for 5 minutes to 0.5 hours. 
     
     
         4 . The method of  claim 1 , wherein the stem cells are from bone marrow, adipose tissue, muscle tissue, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal skin, adolescent skin, or blood. 
     
     
         5 . The method of  claim 1 , wherein the stem cells are autologous, allogeneic or xenogeneic. 
     
     
         6 . The method of  claim 1 , wherein the stem cells are embryonic, post-natal or adult stem cells. 
     
     
         7 . The method of  claim 1 , wherein the stem cells comprise mesenchymal stem cells. 
     
     
         8 . The method of  claim 1 , wherein the stem cells comprise autologous bone marrow aspirate. 
     
     
         9 . The method of  claim 8 , wherein the bone marrow aspirate is drawn intraoperatively. 
     
     
         10 . The method of  claim 8 , wherein cells in the bone marrow aspirate are isolated or concentrated. 
     
     
         11 . The method of  claim 1 , wherein the active agents can regulate cellular growth and/or differentiation, and are selected from the group consisting of small molecules, peptides, growth factors, cytokines, ligands, hormones and combinations thereof. 
     
     
         12 . The method of  claim 1 , wherein the active agents are selected from the group consisting of transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), bone morphogenic protein (BMP), insulin growth factor (IGF), interleukin-I (IL-I), interleukin-11 (IL-11), simvastatsin, dexamethasone, oxysterol, sonic hedgehog, interferon, tumor necrosis factor, nerve growth factor (NGF), fibronectin, RGD peptide, integrin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, osteogenic protein, and combinations thereof. 
     
     
         13 . The method of  claim 1 , wherein the active agents are selected from the group consisting of BMP-2, TGF-beta3, PSGF-AB, PDGF-BB, FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6, FGF-8, IL-11, simvastatsin, dexamethasone, oxysterols, sonic hedgehog, and combinations thereof. 
     
     
         14 . The method of  claim 1 , wherein the active agents include TGF-β and FGFb. 
     
     
         15 . The method of  claim 14 , wherein the active agents include PDGF. 
     
     
         16 . The method of  claim 1 , wherein the active agents are in solution. 
     
     
         17 . The method of  claim 16 , wherein the activated stem cells are separated from the active agents by a procedure comprising filtration, gel filtration, tangential flow filtration, immunoprecipitation, immuno-absorption, affinity chromatography, column chromatography or a combination thereof. 
     
     
         18 . The method of  claim 1 , wherein the active agents are attached to a solid support. 
     
     
         19 . The method of  claim 18 , wherein at least some of the active agents are attached to a solid support via a peptide, an antibody, a chemical cross linker, or a combination thereof. 
     
     
         20 . The method of  claim 1 , wherein the bone graft substitute comprises a calcium salt. 
     
     
         21 . The method of  claim 20 , wherein the calcium salt comprises monocalcium phosphate monohydrate, α-tricalcium phosphate, β-tricalcium phosphate, calcium carbonate, or a combination thereof. 
     
     
         22 . The method of  claim 20 , wherein the bone graft substitute further comprises demineralized bone, a sodium phosphate salt, a polymer or a combination thereof. 
     
     
         23 . The method of  claim 22 , wherein the polymer is collagen, gelatin, hyaluronic acid, a hyaluronate salt, hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose (HEC), xantham gum, guar gum, alginate or a combination thereof. 
     
     
         24 . The method of  claim 1 , wherein the method increases expression of alkaline phosphatase and/or a bone morphogenetic protein (BMP) receptor subunit in the stem cells. 
     
     
         25 . The method of  claims 1 , further comprising implanting the implant composition into a patient. 
     
     
         26 . An implant composition prepared by the method of  claim 1 . 
     
     
         27 . A method for treating a bone injury, disorder or condition in a subject comprising administering the implant composition of  claim 26  to a site of the bone injury, disorder or condition in the subject. 
     
     
         28 . The method of  claim 27 , wherein the bone injury, disorder or condition comprises a broken bone, a bone defect, a bone transplant, a bone graft, bone cancer, a joint replacement, a joint repair, a bone fusion, a bone facet repair, bone degeneration, a dental implants, a dental repair, arthritis, bone reconstruction, or a combination thereof. 
     
     
         29 . A device for activating stem cells comprising a solid support and at least one active agent that promotes differentiation of stem cells into osteogenic cells or osteogenic precursor cells, and the device is adapted: (i) for incubating the stem cells with the at least one active agent, and (ii) for separating the at least one active agent from the stem cell after the incubating step (i). 
     
     
         30 . The device of  claim 29 , wherein the at least one active agent is selected from the group consisting of transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), bone morphogenic protein (BMP), insulin growth factor (IGF), interleukin-I (IL-I), interleukin-11 (IL-11), simvastatsin, dexamethasone, oxysterol, sonic hedgehog, interferon, tumor necrosis factor, nerve growth factor (NGF), fibronectin, RGD peptide, integrin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, osteogenic protein, and combinations thereof. 
     
     
         31 . The device of  claim 29 , wherein the at least one active agent is selected from the group consisting of BMP-2, TGF-beta3, PSGF-AB, PDGF-BB, FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6, FGF-8, IL-11, simvastatsin, dexamethasone, oxysterols, sonic hedgehog, and combinations thereof. 
     
     
         32 . The device of  claim 29 , wherein the at least one active agent includes TGF-β and FGFb. 
     
     
         33 . The device of  claim 32 , wherein the at least one active agent further includes PDGF. 
     
     
         34 . The device of  claim 29 , wherein the at least one active agent is attached to the solid support. 
     
     
         35 . The device of  claim 29 , wherein the solid support comprises a column matrix material, a filter, an culture plate, tube or dish, a flask, a microtiter plate, a bead, a disk, or a combination thereof. 
     
     
         36 . The device of  claim 29 , wherein the solid support comprises plastic, cellulose, cellulose derivatives, magnetic particles, nitrocellulose, glass, fiberglass, latex, or a combination thereof. 
     
     
         37 . The device of  claim 29 , wherein the at least one active agent is in solution. 
     
     
         38 . The device of  claim 29 , wherein the solid support comprises a container. 
     
     
         39 . The device of  claim 29 , wherein the solid support comprises a filter. 
     
     
         40 . The device of  claim 39 , wherein the filter retains cells and bone graft substitute materials but allows passage of the at least one active agent. 
     
     
         41 . The device of  claim 39 , wherein the filter retains the at least one active agent but allows passage of the stem cells. 
     
     
         42 . The device of  claim 29 , wherein the solid support comprises an affinity matrix to remove the at least one active agent. 
     
     
         43 . The device of  claim 29 , wherein the solid comprises streptavidin, biotin, a crosslinker, an antibody or peptide that binds at least one active agent. 
     
     
         44 . The device of  claim 29 , wherein the solid support material does not bind or adversely interact with stem cells. 
     
     
         45 . The device of  claim 29 , wherein the stem cells are from bone marrow, adipose tissue, muscle tissue, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal skin, adolescent skin, or blood. 
     
     
         46 . The device of  claim 29 , wherein the stem cells comprise mesenchymal stem cells. 
     
     
         47 . The device of  claim 29 , wherein the stem cells comprise autologous bone marrow aspirate. 
     
     
         48 . The device of  claim 29 , further comprising a timer for controlling the time for incubating the stem cells with the at least one active agent. 
     
     
         49 . The device of  claim 48 , wherein the timer triggers separation of the at least one active agent from the stem cell after the incubating step (i). 
     
     
         50 . The device of  claim 48 , wherein the timer controls the time for incubating the stem cells with the at least one active agent to 24 hours or less. 
     
     
         51 . The device of  claim 48 , wherein the timer controls the time for incubating the stem cells with the at least one active agent to 5 minutes to 1 hour. 
     
     
         52 . The device of  claim 48 , wherein the timer controls the time for incubating the stem cells with the at least one active agent to 5 minutes to 0.5 hours.