WO2007056433A2 - Procedes de traitement de defauts tissulaires - Google Patents

Procedes de traitement de defauts tissulaires Download PDF

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Publication number
WO2007056433A2
WO2007056433A2 PCT/US2006/043443 US2006043443W WO2007056433A2 WO 2007056433 A2 WO2007056433 A2 WO 2007056433A2 US 2006043443 W US2006043443 W US 2006043443W WO 2007056433 A2 WO2007056433 A2 WO 2007056433A2
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Prior art keywords
stem cells
bmp
bone
site
defect
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PCT/US2006/043443
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English (en)
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WO2007056433A3 (fr
Inventor
Bruce Simon
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Ebi, L.P.
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Priority claimed from US11/268,696 external-priority patent/US7744869B2/en
Priority claimed from US11/268,699 external-priority patent/US20070105769A1/en
Application filed by Ebi, L.P. filed Critical Ebi, L.P.
Publication of WO2007056433A2 publication Critical patent/WO2007056433A2/fr
Publication of WO2007056433A3 publication Critical patent/WO2007056433A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N2/00—Magnetotherapy
    • A61N2/002—Magnetotherapy in combination with another treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
    • A61N1/205—Applying electric currents by contact electrodes continuous direct currents for promoting a biological process
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/40—Applying electric fields by inductive or capacitive coupling ; Applying radio-frequency signals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

Definitions

  • the present invention relates to methods for treating trauma, disease, or tissue disorders.
  • bone remodeling and healing There are a number of complex steps and processes that are involved in the body's response to a tissue defect, such as bone remodeling and healing.
  • One step in the healing of bone defects includes the proliferation of mesenchymal stem cells from the bone marrow, periosteum, and surrounding soft tissue. And unlike most other tissues which heal when injured by forming connective tissue, bone heals by the formation of new bone. Bone is also subject to constant breakdown and re-synthesis in a complex process mediated by osteoblasts, which produce new bone, and osteoclasts, which destroy bone. Osteoblasts arise when mesenchymal stem cells located near bony surfaces differentiate under the influence of growth factors. For example, locally produced bone rnorphogenic protein (BMP) growth factors can transform stem cells into osteoprogenitor cells. Further differentiation of these cells is an important part of bone repair.
  • BMP bone rnorphogenic protein
  • Growth factors are proteins that can affect proliferation and differentiation of various cell types, including stem cells such as mesenchymal stem cells. Certain growth factors have shown clinical benefit in treatment of bone defects, injuries, disorders, or diseases. In particular, the bone morphogenic proteins, including
  • BMP-2 and BMP-7 have shown clinical benefit in the treatment of bone fractures and spine fusions.
  • the amount of the bone morphogenic protein required for treatment is large, often requiring several milligrams, such that clinical use is limited by great expense.
  • Electromagnetic fields can also affect bone repair. Exposure to electric fields can promote connective tissue repair and can accelerate the healing of bone fractures, extracellular matrix synthesis, and the incorporation of bone grafts. For example, the synthesis of cartilage molecules is enhanced by electric stimulation. And furthermore, electric fields can accelerate cell differentiation and stimulate expression of growth factors. [0005] Another response to a bodily trauma, disease, or disorder is the recruitment of pluripotent stem cells to the site of the trauma, disease or disorder. Repair and/or healing are achieved in part by subsequent proliferation and differentiation of the stem cells into specific cells. For example, mesenchymal stem cells proliferate and differentiate into specific cells to repair damage to bone.
  • Stem cells can be obtained from embryonic or adult tissues of humans or other animals. Irrespective of origin, all stem cells are initially unspecialized cells capable of dividing and renewal, and can give rise by differentiation to specialized cell types (see e.g., National Institutes of Health, Stem Cell Basics, available on the internet at http://stemcells.nih.gov/info/basics/). For example, mesenchymal stem cells, which are stem cells obtained from adult or embryonic connective tissues, can differentiate into many different cell types such as bone, cartilage, fat, ligament, muscle and tendon.
  • therapies involving the alteration of cell or tissue properties by exposure to electromagnetic fields have been proposed. See, Aaron and Ciombor, J. Cellular Biochemistry 52: 42-46, 1993; Aaron et al., J. Bone Miner. Res. 4: 227-233, 1989; Aaron and Ciombor, J. Orthop. Res. 14: 582-589, 1996; Aaron et al., Bioelectromagnetics 20: 453-458, 1999; Aaron et al, J. Orthop. Res. 20: 233-240, 2002; Ciambor et al., J.
  • aspects of the present invention provide for using electric or electromagnetic field stimulation, such as pulsed electromagnetic fields (PEMFs) and growth factors to treat a tissue defect in a human or other mammal subject.
  • the effect of the growth factor treatment is potentiated by the electric field. That is, the effect of the growth factor in addition to the electric field is greater than the effect of either the growth factor or the electric field alone.
  • certain embodiments of the present invention can allow for effective treatment using a growth factor and an electric field where the growth factor is at a subefficacious level.
  • the electric stimulation can be a direct current electric field, a capacitatively coupled electric field, or a pulsed electromagnetic fields (PEMF).
  • the growth factor is a member of the transforming growth factor beta (TGF- ⁇ ) superfamily, such as a bone morphorgenic protein (BMP).
  • the present invention provides methods for the treatment of a human or other mammal subject having a tissue defect, by optionally implanting stem cells into the subject at the site of a defect, and administering electric stimulation and growth factor to the stem cells in situ.
  • the present invention provides for modulating stem cell activity using electrical stimulation and growth factors where the result is more efficacious than either administering electrical stimulation or growth factors independently of one another.
  • the present invention provides for modulating stem cell activity using electrical stimulation and growth factors where the amount of growth factor required to achieve a beneficial result is less than the amount required to obtain a beneficial result without electrical stimulation.
  • such benefits can include one or more of: increased or accelerated proliferation of endogenous stem cells in situ; enhanced differentiation of endogenous stem cells in situ; reduced amounts of growth factors required to achieve beneficial effects; increased rate of healing of tissue defects; and more complete healing of tissue defects.
  • the words “preferred” and “preferably” refer to embodiments of the invention that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention. [0018] As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this invention.
  • the present invention involves the treatment of tissue defects in humans or other animal subjects.
  • Specific materials to be used in the invention must, accordingly, be pharmaceutically acceptable.
  • a “pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • the term "electric stimulation” as used herein includes exposing tissue to an electric field, such as a direct current electric field, a capacitatively coupled electric field, an electromagnetic field, a pulsed electromagnetic field (PEMF), or combinations thereof.
  • an electric field such as a direct current electric field, a capacitatively coupled electric field, an electromagnetic field, a pulsed electromagnetic field (PEMF), or combinations thereof.
  • PEM pulsed electromagnetic field
  • the term “electric stimulation,” as used herein, does not include an electric or electromagnetic field associated with ambient conditions, such as an electric field generated by casual exposure to radios, telephones, desktop computers or similar devices.
  • the strength of the electric field produced during electrical stimulation is preferably at least about 0.5 microvolts per centimeter.
  • the current is preferably a direct current of at least about 0.5 microamperes.
  • the electric stimulation comprises administration of direct current, where the direct current electric field has an intensity of at least about 0.5 microamperes, preferably from about 10 to about 200, and preferably from about 20 to about 100 microamperes. . Specific embodiments include those wherein the intensity is about 20, about 60, and about 100 microamperes.
  • the field may be constant, or varying over time. In various embodiments, the field is a temporally varying capacitatively coupled field.
  • a sinusoidally-varying electric field is used.
  • various embodiments use a sinusoidally-varying electric field for electrodes placed across tissue at the site of a tissue defect.
  • Such an implantation site can be a human limb, for example.
  • a sinusoidally-varying electric field has a peak voltage across electrodes placed across the cells of from about 1 volt to about 10 volts, more preferably about 5 volts.
  • the electric field has peak amplitude of from about 0.1 millivolt per centimeter (mV/cm) to about 100 mV/cm, and more preferably about 20 mV/cm.
  • the sinusoidal field has a frequency of from about 1,000 Hz to about 200,000 Hz, preferably about 60,000 Hz.
  • Exposure of tissue defects to PEMFs can be accomplished using methods and apparatus known in the art for exposure of cells and tissues to pulsed fields, for example, as disclosed in the following literature: Binderman et al., Biochimica et Biophysica Acta 844: 273-279, 1985; Aaron et al, Journal of Bone and Mineral Research 4: 227-233, 1989; and Aaron et al., Journal of Orthopaedic Research 20: 233-240, 2002. Parameters of exposure to an electric stimulation field, such as pulse duration, pulse intensity, and numbers of pulses can be determined by a user.
  • pulse duration of a PEMF can be from about 10 microseconds per pulse to about 2000 microseconds per pulse, and is preferably about 225 microseconds per pulse.
  • pulses are comprised in electromagnetic "bursts."
  • a burst can comprise from 1 pulse up to about 200 pulses.
  • a burst comprises from about 10 pulses to about 30 pulses, more preferably about 20 pulses.
  • Bursts can be repeated while applying PEMFs.
  • bursts can be repeated at a frequency of from about 1 Hertz (Hz) to about 100 Hz, preferably at a frequency of about 10 Hz to about 20 Hz, more preferably at a frequency of about 15 Hz.
  • bursts can repeat at a frequency of about 1.5 Hz, or about 76 Hz.
  • a burst can have a duration from about 10 microseconds up to about 40,000 microseconds; preferably, a burst can have a duration of about 4.5 milliseconds.
  • An electric field used herein can be produced using any suitable method and apparatus, including such methods and apparatuses known in the art. Suitable apparatus include a capacitatively coupling device such as a SpinalPak ® (EBI, L.P., Parsippany, New Jersey, U.S.A.) or a DC stimulation device such as an SpF ® XL lib spinal fusion stimulator (EBI, L.P.).
  • a PEMF can be produced using any known method and apparatus, such as using a single coil or a pair of Helmholtz coils.
  • such an apparatus includes the EBI Bone Healing System ® Model 1026 (EBI, L.P.).
  • the electrical stimulation comprises direct current electric field generated using any known device for generating a direct current electric field, such as, for example, an OsteogenTM implantable bone growth stimulator (EBI, L.P., Parsippany, N.J.).
  • growth factor includes at least one growth factor or a mixture of more than one growth factor.
  • the growth factor can be a member of the transforming growth factor beta (TGF- ⁇ ) superfamily.
  • TGF- ⁇ transforming growth factor beta
  • Various embodiments of the present invention can include one or more growth factors selected from: vascular endothelial growth factors (VEGF) such as VEGF-I, a fibroblast growth factor (FGF) such as FGF-2, epidermal growth factor (EGF), vascular endothelial growth factors (VEGF) such as insulin-like growth factors (IGF) such as IGF-I and IGF-II, transforming growth factors (TGF) such as TGF- ⁇ , platelet-derived growth factors (PDGF), EGM, and bone morphogenetic proteins (BMP) such as BMP-2, BMP-4, BMP-6 and BMP-7.
  • VEGF vascular endothelial growth factors
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • VEGF
  • the combination of electric stimulation and growth factor can allow the use of a lower amount of growth factor, where the lower amount of growth factor alone would not obtain an appreciable beneficial result in the absence of the electric stimulation.
  • methods include administering an electric field and a subefficacious amount of growth factor.
  • a "subefficacious amount" of growth factor is an amount of growth factor that is not efficacious in treating a tissue defect by itself.
  • a BMP is administered at an amount from about 1 ng/mL to about 100 ng/mL. Additional embodiments include BMP-2 from about 1 ng/mL to about 100 ng/mL. More preferably, various embodiments include BMP-2 from about 10 ng/mL to about 70 ng/mL, and in one embodiment at a level of about 40 ng/mL.
  • growth factors are administered to the site of a tissue defect at a level less than the levels that such factors are used clinically without electrical stimulation, preferably of from about 0.5% to about 50% of such clinical levels, more preferably from about 1% to about 30%, from about 2% to about 20%, or from 5% to about 10% of the clinical dose without electrical stimulation.
  • a growth factor is administered at a total dose level of from about 0.1 to about 5 mg, preferably from about 0.2 to about 2mg.
  • the present invention provides various embodiments for administration of electric stimulation and growth factors to stem cells in situ, where the electrical stimulation potentiates activity of growth factors.
  • Patentiates or “potentiation” of the effect of the growth factors refers to the ability of the electric field to enhance or increase the effect of growth factors at a particular growth factor concentration, where the growth factors at the same concentration would have a reduced or negligible effect on the stem cells in the absence of the electric field.
  • the methods of the present invention can optionally use endogenous and/or exogenous stem cells.
  • the stem cells are mammalian stem cells and preferably human stem cells.
  • Stem cells can be derived from any known tissue source, for example, cartilage, fat, bone tissue (such as bone marrow), ligament, muscle, synovia, tendon, umbilical cord (such as umbilical cord blood), and embryos.
  • mesenchymal stem cells are used.
  • implanted stem cells are cultured, such as stem cells cultured in the presence of electrical stimulation as disclosed in U.S. Patent Application Serial No. 10/924,241, Simon, filed August 20, 2004, and U.S. Patent Application Serial No.
  • implanted stem cells may be obtained by other means, such as by collection from a human or other animal source.
  • the stem cells may be autologous, that is, collected from the subject to whom they are to be administered.
  • the stem cells may be isolated and concentrated using a variety of methods, including centrifugation, enzymatic, ultrasonic, and other methods known in the art.
  • electrical stimulation is applied to endogenous stem cells at the site of the tissue repair.
  • the stem cells of the invention are mesenchymal stem cells.
  • the stem cells may be mesenchymal stem cells capable of differentiating into bone, cartilage, vasculature, or blood cells.
  • the mesenchymal stem cells can be osteogenic stem cells, chondrogenic stem cells, angiogenic stem cells, or hematopoietic stem cells.
  • the mesenchymal stem cells are embryonic or adult mesenchymal stem cells derived from embryonic or adult tissues, respectively, wherein "adult stem cells” include stem cells established from any post-embryonic tissue irrespective of donor age.
  • the stem cells are autologous stem cells.
  • the stem cells are allogeneic stem cells.
  • the stem cells are xenogeneic stem cells.
  • the stem cells are autologous stem cells or allogeneic stem cells. More preferably, the stem cells are autologous mesenchymal stem cells or allogeneic mesenchymal stem cells. Most preferably, the stem cells are autologous mesenchymal stem cells.
  • Stem cells utilized in the present invention can be collected, established into cell lines, cultured and propagated in vitro by methods including standard methods among those known in the art. Such methods include those disclosed in U.S. Patent 6,355,239, Bruder et al., issued March 12, 2002; and U.S. Patent 6,541,024, Kadiyala et al, issued April 1, 2003.
  • the stem cells of this invention additionally can also be grown in vitro in a culture medium comprising one or more growth factors, such as VEGF-I, a fibroblast growth factor (FGF) such as FGF-2, epidermal growth factor (EGF), an insulin-like growth factor- 1 (IGF) such as IGF- lor IGF-II, a transforming growth factor (TGF) such as TGF- ⁇ , platelet-derived growth factor (PDGF), EGM, and a bone morphogenetic protein (BMP) such as BMP-2, BMP-4, BMP-6 or BMP-7.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • IGF insulin-like growth factor- 1
  • TGF transforming growth factor
  • PDGF platelet-derived growth factor
  • BMP bone morphogenetic protein
  • the stem cells are cultured in a medium comprising growth factors while in the presence of electric stimulation, such as PEMF, and the cultured cells are then implanted at the site of a tissue defect and further subjected to electric stimulation, such as PEMF.
  • electric stimulation such as PEMF
  • the present invention of administering electric stimulation and growth factor can, in various embodiments, modulate endogenous and/or exogenous stem cell activity.
  • modulate refers to the modification of one or more activities of stem cells by, for example, enhancing or increasing such activities.
  • the activity is one or more of: increased proliferation, enhanced production of molecules normally produced by the stem cells (such as molecular components of the extracellular matrix (ECM)), and accelerated differentiation of stem cells into differentiated cell types.
  • Accelerated differentiation of stem cells includes enhanced production of differentiation markers of differentiated cell types derived from the stem cells, such as specialized ECM markers, wherein "enhanced production” includes increased and/or accelerated production of such markers, as compared to control stem cells that are not subjected to an electric or electromagnetic field but otherwise are under the same conditions.
  • the present invention provides methods of treating a human or other mammal subject having a tissue defect, by administering electrical stimulation and growth factors at the site of a tissue defect in a subject.
  • such methods involve treatment of a tissue defect with electrical stimulation and growth factors, using less growth factor than treatment without electrical stimulation. Accordingly, in preferred embodiments, such methods involve treatment of a tissue defect by using a subefficacious amount of growth factor in conjunction with electrical stimulation.
  • tissue defects include any condition involving tissue which is inadequate for physiological or cosmetic purposes.
  • defects include those that are congenital, those that result from or are symptomatic of disease or trauma, and those that are consequent to surgical or other medical procedures.
  • Embodiments include treatment for vascular, bone, skin, nerve, and organ tissue defects. Examples of such defects include those resulting from osteoporosis, spinal fixation procedures, hip and other joint replacement procedures, chronic wounds, myocardial infarction, fractures, sclerosis of tissues and muscles, Alzheimer's disease, Parkinson's disease, and spinal cord or other nerve injury.
  • the compositions and methods of this invention may be used to repair bone or cartilage defects.
  • bone defects include any condition involving skeletal tissue which is inadequate for physiological or cosmetic purposes.
  • defects include those that are congenital (including birth defects), those that result from disease or trauma, and those that are consequent to surgical or other medical procedures.
  • defects include those resulting from bone fractures (such as hip fractures and spinal fractures), osteoporosis, spinal fixation procedures, intervertebral disk degeneration (e.g., herniation), and hip and other joint replacement procedures.
  • the present invention provides methods of treating a human or other mammal subject having a tissue defect by implanting stem cells treated with electrical stimulation and growth factors at the site of the tissue defect.
  • methods comprise administering electric stimulation and growth factors to modulate endogenous stem cells at a tissue defect site.
  • stem cells are implanted in a culture medium in which they are grown. In other embodiments, stem cells are isolated from a culture medium, and implanted.
  • a pharmaceutically-acceptable scaffold is implanted in the human or mammal subject at the site of the tissue defect.
  • a "scaffold" is a material that contains or supports tissue growth, preferably enabling growth at the site of implantation.
  • stem cells, growth factors or combinations thereof can be mixed with a scaffold material prior to implantation.
  • a scaffold material is implanted either before or after the stem cells are implanted.
  • Suitable scaffold materials include porous or semi-porous, natural, synthetic or semi-synthetic materials.
  • a scaffold material can be an osteoconductive material.
  • Scaffold materials include those selected from the group consisting of bone (including cortical and cancellous bone), demineralized bone, ceramics, polymers, and combinations thereof. Ceramics include any of a variety of ceramic materials known in the art for use for implanting in bone, including calcium phosphate (including tricalcium phosphate, tetracalcium phosphate, hydroxyapatite, and mixtures thereof. Polymers include collagen, gelatin, polyglycolic acid, polylactic acid, polypropylenefumarate, and copolymers or combinations thereof. Ceramics useful herein include those described in U.S. Patent 6,323,146 to Pugh et al., issued November 27, 2001, and U.S.
  • Patent 6,585,992 to Pugh et al. issued July 1, 2003.
  • a preferred ceramic is commercially available as ProOsteonTM from Interpore Cross International, Inc. (Irvine, California, U.S.A.).
  • Growth factors are preferably incorporated into such composition at levels below those known in the art for compositions used without electrical stimulation.
  • growth factors are present at a concentration of from about 0.01 mg/ml to about 1 mg/ml of scaffold material, preferably from about 0.08 mg/ml to about 0.5 mg/ml, preferably from about 0.1 mg/ml to about 0.3 mg/ml, of scaffold material.
  • Example 1 In a method for enhancing spinal disc repair, the growth factor
  • BMP-2 suspended in a vehicle at 0.1 mg/mL, is injected into a degenerating spinal disc in a human patient.
  • An external device providing a capacitatively coupled electric field, or a PEMF is then worn by the patient.
  • Exposure of the degenerating spinal disc site to an electric or electromagnetic field produced by the device and conjunctive administration of growth factors stimulates endogenous stem cells to proliferate and differentiate into nucleus cells and annulus disc cells and also increases extracellular matrix production by those cells, leading to disc repair.
  • BMP-4, BMP-6 and BMP-7 are substituted for BMP-2, with substantially similar results.
  • a method for treating a bone fracture about 0.5 mg of BMP-7 is injected into the site of the fracture and a pulsed electromagnetic field (PEMF) is applied using a Helmholtz coil. Radiological imaging of the fracture indicates substantial healing of the fracture after four weeks.
  • PEMF pulsed electromagnetic field
  • bone marrow-derived adult mesenchymal stem cells are mixed with growth factors and osteoinductive granules comprising a calcium phosphate material such as hydroxyapatite, and implanted into a patient.
  • An implantable direct current stimulator is placed internally in the vicinity of the graft to provide an electric field in situ to enhance bone formation. Bone healing is accelerated through this treatment.
  • noninvasive electrical stimulation is effected using an electric or electromagnetic field generating device to apply capacitatively coupled electric fields or PEMFs, with substantially similar results.
  • a composition comprising a scaffold material, such as demineralized bone and/or collagen is implanted with the stem cells and growth factors, with substantially similar results.
  • a hip fracture is treated with administration of an electric field, growth factor, and implantation of exogenous stem cells.
  • a culture system is used to expand mesenchymal stem cell numbers or generate three-dimensional constructs.
  • mesenchymal stem cells are derived from muscle, supplied with medium containing growth factors, and grown in culture dishes placed between pairs of Helmholtz coils to generate a uniform PEMF.
  • the stem cells are then harvested, and mixed with collagen as a scaffold material.
  • the composition is then implanted at the site of the fracture, and electric stimulation is continued in situ, thereby accelerating healing of the bone.
  • the stem cells supplied with medium containing growth factors, are grown in culture dishes placed within a capacitatively coupled electric field, with substantially similar results. Also in the above example, the stem cells are derived from bone marrow, muscle, fat, umbilical cord blood, or placenta, with substantially similar results. Also in the above example, collagen is replaced with polyglycolic acid or polylactic acid, with substantially similar results.
  • Example 5
  • the differentiation of mesenchymal stem cells is enhanced by administration of PEMF and growth factor BMP-2.
  • Human mesenchymal stem cells are plated in culture dishes such as, for example, 10 cm 2 culture dishes, and the non-differentiating cultures are grown to near confluence.
  • the cells in the dishes are then stimulated to undergo osteoblast differentiation in the presence or absence of PEMFs and growth factor BMP-2, where BMP-2 is added to the medium to make 40 ng/mL. Samples are taken at different times throughout the differentiation process and examined. Day of plating is designated as day -2. At day 0 (2 days later), cells are stimulated down the osteoblast differentiation pathway.
  • Osteoblast differentiation (to mineralization in vitro) is induced with osteoblast medium (Mesenchymal Stem Cell Growth Medium/10% Fetal Bovine Serum/0.1 ⁇ M dexamethasone/50 ⁇ M ascorbate/10 mM ⁇ glycerophosphate/50ng/mL BMP-4).
  • Cell numbers and extracts are collected at days 0, 1, 2, 6, 9, 12, 14, 21, and 28 following mineralization stimulus. Some cells are stained for mineralized state, others are used for Western blot, RNA extraction, osteocalcin assays, and alkaline phosphatase assays.
  • stem cells subjected to both PEMF and BMP-2 show increased proliferation compared to stem cells subjected to only BMP-2, as evidenced by increased incorporation of 32 P dCTP, as well as increased differentiation of osteoblasts, as evidenced by increased amounts of osteocalcin and alkaline phosphatase, as well as various osteocalcin mRNAs detected using Northern blot analysis.
  • stem cells treated with PEMF and BMP-2 show increased proliferation compared to control stem cells not subjected to either PEMF or BMP-2.
  • the culture matrix comprising stem cells and BMP-2
  • the culture matrix is mixed with osteoconductive granules, and implanted at the site of a spinal fusion surgery.
  • PEMF is then applied to the fusion site.
  • Radiographic imaging indicates substantial fusion of the vertebra within six weeks.

Abstract

La présente invention concerne des procédés de traitement de défauts tissulaires, comprenant des défauts tels que des fractures osseuses, des fusions de vertèbres et une réparation de disques vertébraux, à l’aide de champs électriques ou électromagnétiques et de facteurs de croissance. Dans divers modes de réalisation, la présente invention propose des procédés pour le traitement d’un sujet humain ou d’un autre sujet mammifère en ayant besoin, par l’administration d’une stimulation électrique et de facteurs de croissance pour stimuler les cellules souches endogènes chez le sujet afin de faciliter la cicatrisation. D’autres modes de réalisation comprennent des procédés d’administration d’une stimulation électrique, de facteurs de croissance et de cellules souches dans le défaut. Dans plusieurs modes de réalisation, la quantité du facteur de croissance est une quantité faible.
PCT/US2006/043443 2005-11-07 2006-11-07 Procedes de traitement de defauts tissulaires WO2007056433A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11/268,696 US7744869B2 (en) 2003-08-20 2005-11-07 Methods of treatment using electromagnetic field stimulated mesenchymal stem cells
US11/268,696 2005-11-07
US11/268,699 2005-11-07
US11/268,699 US20070105769A1 (en) 2005-11-07 2005-11-07 Methods of treating tissue defects

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WO2007056433A2 true WO2007056433A2 (fr) 2007-05-18
WO2007056433A3 WO2007056433A3 (fr) 2007-07-05

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US9770339B2 (en) 2005-07-14 2017-09-26 Stout Medical Group, L.P. Expandable support device and method of use
CN109289119A (zh) * 2018-09-16 2019-02-01 华北理工大学 一种用于脊柱康复系统的磁性纳米粒子球混合物
CN109289118A (zh) * 2018-09-16 2019-02-01 华北理工大学 一种用于脊柱康复系统中的呼吸指示装置
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