US20050106724A1 - Pluripotent embryonic-like stem cells derived from teeth and uses thereof - Google Patents

Pluripotent embryonic-like stem cells derived from teeth and uses thereof Download PDF

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US20050106724A1
US20050106724A1 US10/497,206 US49720605A US2005106724A1 US 20050106724 A1 US20050106724 A1 US 20050106724A1 US 49720605 A US49720605 A US 49720605A US 2005106724 A1 US2005106724 A1 US 2005106724A1
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cells
lineage
stem cells
factor
stem
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Joerg Schierholz
Hans-Florian Zeilhofer
Karl-Heiz Hoffman
Christian Morsczexk
Norbert Brenner
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Stiftung Caesar Center of Advanced European Studies and Research
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Stiftung Caesar Center of Advanced European Studies and Research
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Assigned to STIFTUNG CAESAR reassignment STIFTUNG CAESAR ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHIERHOLZ, JORG, ZEILHOFER, HANS-FLORIAN, HOFFMANN, KARL-HEINZ, BRENNER, NORBERT, MORSCZECK, CHRISTIAN
Publication of US20050106724A1 publication Critical patent/US20050106724A1/en
Priority to US12/068,695 priority patent/US8192987B2/en
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    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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Definitions

  • This invention relates generally to pluripotent stem cells, including embryonic-like pluripotent stem cells derived from teeth.
  • the invention also relates to uses of the stem cells for tissue engineering in cell or tissue transplantation, in gene therapy, and in identifying, assaying or screening with respect to cell-cell interactions, lineage commitment, development genes and growth or differentiation factors.
  • Organs, tissues and their associated connective tissue components assayed to date include whole embryo, whole foetus, skeletal muscle, dermis, fat, tendon, ligament, perichondrium, periosteurn, heart, aorta, endocardium, myocardium, epicardium, large arteries and veins, granulation tissue, peripheral nerves, peripheral ganglia, spinal cord, dura, leptomeninges, trachea, oesophagus, stomach, small intestine, large intestine, liver, spleen, pancreas, parietal peritoneum, visceral peritoneum, parietal pleura, visceral pleura, urinary bladder, gall bladder, kidney associated connective tissues and bone marrow.
  • the invention enables the skilled person to identify, separate and to build teeth like and teeth related organs and tissues by tissue engineering.
  • the surprisingly advantage of the invention is the preferred use of follicle cells of wisdom teeth, which are routinely extracted in adults and children.
  • stem cells were developed which freely available even for any adult patients.
  • these said stem cells are differentiable into a periodontal ligament related biological membrane.
  • a Stem Cell can replicate itself and produce cells that take on more specialized functions.
  • the function adopted by the more differentiated daughter cells and their progeny is commonly referred to as the developmental potential, or potency, of the stem cells.
  • Stem cells that give rise to only one type differentiated cell are termed unipotent.
  • the relative terms oligopotent, multipotent, and pluripotent represent an increase in the number of differentiated types from new to many or most.
  • a totipotent cell is one that can generate the totality of cell types that can comprise the organism. In practice, these few terms poorly describe a continuum of possibilities.
  • pluripotent stem cells a promising resource for tissue engineering and transplantation.
  • tissue engineering and transplantation Through the use of enrichment, selection, expression, and sorting technologies, in vitro differentiation of stem cells will certainly contribute to future transplantation therapies.
  • Multipotent stem cells can be cultured from a number of foetal and adult sources.
  • bone marrow which contains both haematopoietic stem cells (Civin et al., 1984) and mesenchymal stem cells (Pittenger et al., 1999).
  • Neural stem cells have also been cultured from the ependymal cells lining the brain ventricles (Johansson et al., 1999a). It is likely that of the many lineage-restricted stem cell populations that exist in vivo, some will be amenable to in vitro growth and analysis.
  • the organization of the embryo into three layers roughly corresponds to the organization of the adult, with gut on the inside, epidermis on the outside, and connective tissue in between.
  • the endoderm is the source of the epithelial linings of the respiratory passages and gastrointestinal tract and gives rise to the pharynx, oesophagus, stomach, intestine and to many associated glands, including salivary glands, liver, pancreas and lungs.
  • the mesoderm gives rise to smooth muscular coats, connective tissues, and vessels associated with the tissues and organs; mesoderm also forms most of the cardiovascular system and is the source of blood cells and bone marrow, the skeleton, striated muscles, and the reproductive and excretory organs.
  • Ectoderm will form the epidermis (epidermal layer of the skin), the sense organs, and the entire nervous system, including brain, spinal cord, and all the outlying components of the nervous system.
  • Reserve stem cells include progenitor stem cells and pluripotent stem cells.
  • Progenitor cells precursor stem cells, immediate stem cells, and forming or -blast cells, e.g., myoblasts, adipoblasts, chondroblasts, etc.) are lineage-committed.
  • Unipotent stem cells will form tissues restricted to a single lineage (such as the myogenic, fibrogenic, adipogenic, chondrogenic, osteogenic lineages, etc.).
  • Bipotent stem cells will form tissues belonging to two lineages (such as the chondro-osteogenic, adipo-fibroblastic lineages, etc.).
  • Tripotent stem cells will form tissues belonging to three lineages (such as chondro-osteo-adipogenic lineage, etc.).
  • Multipotent stem cells will form multiple celltypes within a lineage (such as the hematopoietic lineage).
  • Progenitorstem cells will form tissues limited to their lineage, regardless of the inductive agent that may bedded to the medium. They can remain quiescent.
  • Lineage-committed progenitor cells are capable of self-replication but have a limited life-span (approximately 50-70 celldoublings) before programmed cell senescence occurs. They can also be stimulated by various growth factors to proliferate. If activated to differentiate, these cells require progression factors (i.e., insulin, insulin-like growth factor-1, andinsulin-like growthfactor-11) to stimulate phenotypic expression.
  • pluripotent cells are lineage-uncommitted, i.e., they are not committed to any particular tissue lineage. They can remain quiescent. They can also be stimulated.
  • progenitor and pluripotent stem cells from the mesodermal germ layer include the unipotent myosatellite myoblasts of muscle; the unipotent adipoblast cells of adipose tissue); the unipotent chondrogenic cells and osteogenic cells of the perichondrium and periosteum, respectively; the bipotent adipofibroblasts of adipose tissue (Vierck et al., 1996); the bipotent chondrogenic/osteogenic stem cells of marrow); the tripotent hondrogenic/osteogenic/adipogenic stem cells of marrow (Pittenger et al., 1999); the multipotent hematopoietic stem cells of marrow; the multipotent cadiogenic/hematopoietic/endotheliogenic cells of marrow; and the pluripotent mesenchymal stem cells of the connective tissues.
  • Pluripotent mesenchymal stem cells can be utilized for the replacement of potentially multiple tissues of mesodermal origin (i.e., bone, cartilage, muscle, adipose tissue, vasculature, tendons, ligaments and hematopoietic), such tissues generated, for instance, ex vivo with specific morphogenetic proteins and growth factors to recreate the lost tissues. The recreated tissues would then be transplanted to repair the site of tissue loss.
  • An alternative strategy could be to provide pluripotent stem cells, as cellular compositions or incorporated, for instance, into matrices, transplant into the area of need, and allow endogenous morphogenetic proteins and growth factors to induce the pluripotent stem cells to recreate the missing histoarchitecture of the tissue.
  • One object of the invention is to solve the problem of teeth- and teeth-related tissue substitution. Another object is to provide a source of regenerative cells from adults.
  • the present invention relates to pluripotent stem cells, particularly to pluripotent, multipotent, tri-bi- and unipotent stem cells.
  • the invention further relates to methods of purifying pluripotent embryonic-like stem cells and to compositions, cultures and clones thereof.
  • the present invention also relates to a method of transplanting the pluripotent stem cells of the present invention in a mammalian host, such as human, comprising introducing the stem cells, into the host.
  • the invention further relates to methods of in vivo administration of a protein or gene of interest comprising transfecting a pluripotent stem cell with a construct comprising DNA which encodes a protein of interest and then introducing the stem cell into the host where the protein or gene of interest is expressed.
  • the present invention also relates to methods of producing mesodermal, endodermal or ectodermal lineage-committed cells by culturing or transplantation of the pluripotent stem cells or segregation these germ layers through progressive lineage-commitment into progenitor (multipotent, tripotent, bipotent and eventually unipotent) lineages of the present invention.
  • the present invention also relates to engineering of teeth and teeth surrounding tissue such as paradentin or bone, vessels or nerves related to teeth.
  • the invention relates also to the development of a stem cell bank for teeth or teeth derived tissues teeth derived tissues, as well as the development of membrane-like meso/endodermal matrices which can be used for a wide range of regenerative medicine.
  • the stem cell of the invention is obtainable from the non-embryonic tissue isolated from the dental follicle of tooth or wisdom tooth which are able to differentiate into a periodontal ligament like membrane structure. Additionally the stem cell of the invention is able to differentiate into a mineralised tissue like structure.
  • the stem cell of the invention which was derived from non-embryonic or postnatal animal cells or tissue is capable of self-renewal and capable of differentiation to cells of endodermal, ectodermal and mesodermal lineages.
  • the stem cell of the invention is a human cell.
  • Subject of the present invention is also a pluripotent mesenchymal stem cell obtainable from the stem cell of the invention.
  • the stem cell of the invention can be kept in a culture comprising:
  • this culture is further comprising a proliferation factor or lineage commitment factor.
  • the stem cells are human cells.
  • Subject of the invention is also a method of isolating an pluripotent embryonic-like stem cell, comprising the steps of
  • a clonal pluripotent embryonic-like stem cell line can be obtained by a method of isolating, comprising the steps of
  • a stem cell of the invention which is genetically engineered to express a gene or protein of interest is also subject matter of the present invention.
  • Such a stem cell may be generated by a method of producing a genetically engineered pluripotent embryonic-like stem cell comprising the steps of
  • the stem cell obtainable by this method is genetically engineered pluripotent embryonic-like stem cell, such as a dental follicle cell.
  • the stem cells of the invention may also be used in a method for detecting the presence or activity of an agent which is a lineage-commitment factor.
  • This method of the invention comprises the steps of
  • the stem cells of the invention may also be used in a method of testing the ability of an agent, compound or factor to modulate the lineage-commitment of a lineage uncommitted cell.
  • This method of the invention comprises
  • the stem cells of the invention may also be used in an assay system for screening agents, compounds or factors for the ability to modulate the lineage-commitment of a lineage uncommitted cell.
  • This method of the invention comprises:
  • the stem cells of the invention may also be used in a method for detecting the presence or activity of an agent which is a proliferation factor.
  • This method of the invention comprises the steps of
  • the stem cells of the invention may also be used in a method of testing the ability of an agent, compound or factor to modulate the proliferation of a lineage uncommitted cell.
  • This method of the invention comprises:
  • Subject matter of the present invention is also a method for culturing stem cells of the invention into membranes which will used for regenerating parodontitis, tendons, ligaments, fascia and other kinds of connecting tissues supporting differentiation to mesothelic/endothelic cells into blood vessels or ectodermal or into-neuronal tissue.
  • the stems cells of the invention can be cultured by a method for culturing in any media containing one or in combination for differentiation: insulin, retinoic acid, indomethacin, isobtylxanthine, theophylline, transforming-growth-factor-beta (any), bone morphogenetic protein (any), Fibroblast growth factor (any), Epidermal Growth factor (any), Platelet derived growth factor (any), Vascular endothelial growth factor (any), hepatocyte growth factor, Interferon (any), Insulin like growth factor (any), Interleukine (any), nerve growth factor.
  • the invention is also related to a method of ex vivo administration of a protein or gene of interest comprising the step of transfecting the pluripotent embryonic-like stem cell of the invention with a vector comprising DNA or RNA which expresses a protein or gene of interest.
  • Subject matter of the present invention is also a method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of pluripotent embryonic-like stem cells, or cells or tissues derived therefrom.
  • a mammal a therapeutically effective amount of pluripotent embryonic-like stem cells, or cells or tissues derived therefrom.
  • the skilled person knows how choose time development stage of the stem cell of the invention in order to implant the stem cell as well as its dosage.
  • the cell number to be applied can vary dependent on the disease to be cured. In animal experiments typically cells were administered in a number about 10 6 cells.
  • the stem cell of the invention can be used for tissue repair or transplantation in mammals by administering the stem cell to a mammal in a therapeutically effective amount of pluripotent embryonic-like stem cells, or cells or tissues derived therefrom.
  • the stem cells can be used in a method of preventing and/or treating cellular debilitations, derangements 2 and/or dysfunctions and/or other disease states in mammals by administering to a mammal a therapeutically effective amount of a mesodermal lineage-committed cell derived from the stem cell of the invention.
  • the stem cells of the invention make possible a method of tissue repair or transplantation in mammals, comprising administering to a mammal a therapeutically effective amount of a mesodermal lineage-committed cell derived from the stem cell of the invention.
  • the stem cells of the invention can be essential part of a pharmaceutical composition for the treatment of cellular debilitation, derangement and/or dysfunction in mammals, comprising:
  • a proliferation factor or lineage-commitment factor is added.
  • the stem cells of the invention can be present in a scaffold, consisting of Table 1 to engineer teeth by culturing stem cells of the invention on matrices or for creating teeth-connection periodontal cell lines for treating periodontal diseases.
  • the scaffold consisting of table 1 can also be used for building teeth-surroundings bones/bone matrix alveolar bone or jaw bone or building arterial and venous vessels in mouth.
  • a stem cell derived from teeth is seeded on an extra cellular matrix controlling its behaviour by providing adhesion signals, and growth factor binding sites seeded on a scaffold having cell binding domain sequences according to table 2 consisting of table 1.
  • the invention also concerns a method of culturing of cells wherein the cells are seeded on matrices in a bioreactor in table 3.
  • the stem cells of the invention can be employed also in a method to implant stem cells in periodontium to rebuild periodontium, to improve healing teeth extraction and skin lesions.
  • stem cells of the invention are implanted in periodontium to build or repair cementum of tooth.
  • the present invention relates to a culture comprising:
  • Such stem cell containing cultures may further comprise a proliferation factor or lineage commitment factor.
  • the stem cells of such cultures may be isolated from non-human cells or human cells.
  • the invention further relates to methods of isolating an pluripotent embryonic-like stem cell.
  • a method of isolating an pluripotent embryonic-like stem cell of the present invention comprises the steps of
  • the invention further relates to methods of isolating an pluripotent embryonic-like stem cell,
  • a method of isolating an pluripotent embryonic-like stem cell of the present invention comprises the steps of
  • the invention further relates to methods of isolating an pluripotent embryonic-like stem cell.
  • a method of isolating an pluripotent embryonic-like stem cell of the present invention comprises the steps of
  • the invention further relates to methods of isolating an pluripotent embryonic-like stem cell,
  • a method of isolating an pluripotent embryonic-like stem cell of the present-invention comprises the steps of
  • the methods of isolating an pluripotent embryonic-like stem cell relate to methods whereby a clonal population of such stem cells is isolated, wherein a single pluripotent embryonic-like stem cell is first isolated and then further cultured and expanded to generate a clonal population.
  • a single pluripotent embryonic-like stem cell may be isolated by means of limiting dilution or such other methods as are known to the skilled artisan.
  • the present invention also relates to a clonal pluripotent embryonic-like stem cell line developed by such method.
  • the present invention relates to pluripotent embryonic-like stem cells or populations of such cells which have been transformed or transfected and thereby contain and can express a gene or protein of interest.
  • this invention includes pluripotent embryonic-like stem cells genetically engineered to express a gene or protein of interest.
  • the present invention further encompasses lineage-committed cells, which are derived from a genetically engineered pluripotent embryonic-like stem cell, and which express a gene or protein of interest.
  • the lineage-committed cells may be endodermal, ectodermal or mesodermal lineage-committed cells and may be pluripotent, such as a pluripotent mesenchymal stem cell, or progenitor cells, such as an adipogenic or a myogenic cell.
  • the invention then relates to methods of producing a genetically engineered pluripotent embryonic-like stem cell comprising the steps of
  • the present invention encompasses genetically engineered pluripotent embryonic-like stem cell(s), including human and non-human cells, produced by such method.
  • the present invention further relates to methods for detecting the presence or activity of an agent which is a lineage-commitment factor comprising the steps of
  • the present invention also relates to methods of testing the ability of an agent, compound or factor to modulate the lineage-commitment of a lineage uncommitted cell which comprises
  • the invention includes an assay system for screening of potential agents, compounds or drugs effective to modulate the proliferation or lineage-commitment of the pluripotent embryonic-like stem cells of the present invention.
  • the present invention relates to an assay system for screening agents, compounds or factors for the ability to modulate the lineage-commitment of a lineage uncommitted cell, comprising:
  • the invention also relates to a method for detecting the presence or activity of an agent which is a proliferation factor comprising the steps of.
  • the invention includes methods of testing the ability of an agent, compound or factor to modulate the proliferation of a lineage uncommitted cell which comprises
  • the invention further relates to an assay system for screening agents, compounds or factors for the ability to modulate the proliferation of a lineage uncommitted cell, comprising:
  • the assay system could importantly be adapted to identify drugs or other entities that are capable of modulating the pluripotent embryonic-like stem cells of the present invention, either in vitro or in vivo.
  • Such an assay would be useful in the development of agents, factors or drugs that would be specific in modulating the pluripotent embryonic-like stem cells to, for instance, proliferate or to commit to a particular lineage or cell type.
  • drugs might be used to facilitate cellular or tissue transplantation therapy.
  • the assay system(s) could readily be adapted to screen, identify or characterize genes encoding proliferation or lineage-commitment factors or encoding proteins or molecules otherwise involved in cellular differentiation and development.
  • genes encoding proteins involved in or expressed during differentiation along a particular lineage could be identified by known methods (for instance cDNA libraries, differential display, etc.
  • the pluripotent embryonic-like stem cells of the present invention could be cultured under conditions giving rise to a particular lineage and the genes therein expressed then characterized.
  • Factors and proteins necessary for maintaining the pluripotent embryonic-like stem cells of the present invention in a pluripotent embryonic-like state might also be similarly identified and characterized by culturing the pluripotent embryonic-like stem cells of the present invention under conditions maintaining their self-renewal capacity and characterizing the genes and proteins so expressed or which, when provided exogenously, will maintain the self-renewal capacity.
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the pluripotent embryonic-like stem cells of the present invention, including cells or tissues derived therefrom, or upon, agents or other drugs determined to act on any such cells or tissues, including proliferation factors and lineage-commitment factors.
  • One exemplary therapeutic method is associated with the prevention or modulation of the manifestations of conditions causally related to or following from the lack or insufficiency of cells of a particular lineage, and comprises administering the pluripotent embryonic-like stem cells of the present invention, including cells or tissues derived therefrom, either individually or in mixture with proliferation factors or lineage-commitment factors in an amount effective to prevent the development or progression of those conditions in the host.
  • the present invention includes therapeutic methods, including transplantation of the pluripotent embryonic-like stem cells of the present invention, including lineage-uncommitted populations of cells, lineage-committed populations of cells, tissues and organs derived there from, in treatment or alleviation of conditions, diseases, disorders, cellular debilitations or deficiencies which would benefit from such therapy.
  • These methods include the replacement or replenishment of cells, tissues or organs. Such replacement or replenishment may be accomplished by transplantation of the pluripotent embryonic-like stem cells of the present invention or by transplantation of lineage-uncommitted populations of cells, lineage-committed population of cells, tissues or organs derived therefrom.
  • the present invention includes a method of transplanting pluripotent embryonic-like stem cells in a host comprising the step of introducing into the host the pluripotent embryonic-like stem cells of the present invention.
  • this invention provides a method of providing a host with purified pluripotent embryonic-like stem cells comprising the step of introducing into the host the pluripotent embryonic-like stem cells of the present invention.
  • this invention includes a method of in vivo administration of a protein or gene of interest comprising the step of transfecting the pluripotent embryonic-like stem cells of the present invention with a vector comprising DNA or RNA which expresses a protein or gene of interest.
  • the present invention provides a method of tissue repair or transplantation in mammals, comprising administering to a mammal a therapeutically effective amount of pluripotent embryonic-like stem cells.
  • the present invention provides a method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of pluripotent embryonic-like stem cells.
  • the present invention provides a method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of a endodermal, ectodermal or mesodermal lineage-committed cell derived from the pluripotent embryonic-like stem cells of the present invention.
  • the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise proliferation factors or lineage-commitment factors, alone or in combination with the pluripotent embryonic-like stem cells of the present invention, or cells or tissues derived therefrom, or other similarly effective agents, drugs or compounds identified for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • compositions for use in therapeutic methods which comprise or are based upon the pluripotent embryonic-like stem cells of the present invention, including lineage-uncommitted populations of cells, lineage-committed populations of cells, tissues and organs derived therefrom, along with a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising proliferation factors or lineage commitment factors that act on or modulate the pluripotent embryonic-like stem cells of the present invention and/or the cells, tissues and organs derived therefrom, along with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of proliferation factors or lineage commitment factors may be further comprise the pluripotent embryonic-like stem cells of the present invention, or cells, tissues or organs derived therefrom.
  • the pharmaceutical compositions may comprise the pluripotent embryonic-like stem cells of the present invention, or cells, tissues or organs derived therefrom, in apolymeric carrier or extracellular matrix.
  • compositions for the treatment of cellular debilitation, derangement and/or dysfunction in mammals comprising:
  • compositions of the present invention also include compositions comprising endodermal, ectodermal or mesodermal lineage-committed cell(s) derived from the pluripotent embryonic-like stem cells of the present invention, and a pharmaceutically acceptable medium or carrier. Any such pharmaceutical compositions may further comprise a proliferation factor or lineage-commitment factor.
  • the origin of the tooth is the tooth germ with the ability to form all cells building a tooth.
  • one has a stage of pluripotency, a undifferentiated precursor stage, with the capability to differentiate to cells of the dental organ, dental papilla and dental follicle.
  • FIG. 11 explains the development of a tooth (From: http//:bite-it.helsinki.fl).
  • First arch epithelium loses this odontogenic potential, which is then assumed by the ectomesenchyme so that it can elicit tooth formation from a variety of epithelia.
  • recombination of late first arch ectomesenchyme with embryonic plantar (foot) epithelium changes the development direction of the epithelium so that a dental organ is formed
  • the epithelial dental organ is recombined with skin mesenchyme, it loses its dental characteristics and assumes those of epidermis.
  • the cells assume a cuboidal shape and form the external, or outer, dental epithelium.
  • the cells bordering on the dental papilla differentiate into two histologically distinct components. Those immediately adjacent to the dental papilla assume a short columnar shape and are characterized by high glycogen content; together they form the internal (or inner) dental epithelium. Between the internal dental epithelium and the newly differentiated stellate reticulum some epithelial cells differentiate into a layer called the stratum intermedium. The cells of this layer are soon characterized by an exceptionally high activity of the enzyme alkaline phosphatase.
  • both layers should be considered as a single function unit responsible for the formation of enamel.
  • the internal dental epithelium meets the external dental epithelium at the rim of the dental organ; this junctional zone is known as the cervical loop.
  • Periodontal ligament is a connective tissue with fibroblasts, collagen and vascular channels and blood vessels which defined a membrane like structure.
  • tissue pieces were incubated in a 0.250/0 trypsin in D-MEM without Calcium supplemented with EDTA for up to 18 h for a maximal penetration with little trypsin activity, Trypsin were decant and discard and tissue were treated with residual trypsin at 37° C. for 20 min-30 min in phosphate buffer.
  • Single-cell suspensions were obtained by passing the cells through a 70- ⁇ m strainer (Falcon). Bone marrow cells, processed from marrow aspirates of normal human adult volunteers were purchased from Poietic Technologies and then washed in growth medium.
  • Knockout D-MEM serumreplacement media (GIBCO/BRL) supplemented with 2 mM L-glutamine/100 units/ml penicillin/100 ⁇ g/ml streptomycin (Biofluids, Rockville, Md.) or DMEM (Biochrom) supplemented with 20% FCS (Equitech-Bio, Kerrville, Tex.)/100 ⁇ M L-ascorbic acid 2-phosphate (Wako Pure Chemicals, Osaka)/2 mM L-glutamine/100 units/ml penicillin/100 ⁇ g/ml streptomycin (Biofluids, Rockville, Md.) or MSCBM BulletKit (Poietics) and then incubated at 37° C.
  • Bone marrow cells were cultured in MSCBM BulletKit (Poietics). To assess colony-forming efficiency, day 14 cultures were fixed with 4% formalin, and then stained with 0.1% toluidine blue. For the induction of calcified bone matrix deposition in vitro cells were cultured in D-MEM (Biochrom) supplemented with 10% FCS, 2 mM L-Glutamine (sigma), 10-8 M Dexamethason (sigma), 100 ⁇ M L-ascorbic acid 2-phosphate, 1.8 mM KH 2 PO 4 (Sigma), 20 mM HEPES (Sigma).
  • DFSCs Dental follicle Stem cells
  • BMSCs Bone marrow stromal cells
  • Antibodies used Notch Ab-1 (A6) Mouse Mab (anti-Notch1) (NeoMarkers), AC 133/2 (anti-CD133) (Miltenyl), Flk-1 (anti-VEGFR-2), Mouse IgG1 (clone 53) anti mouse FNK (fibroblast growth factor), Mouse IgG1 (clone 25) anti rat Nestin, Mouse IgG1 (clone 52 anti human GFAP (glial fibrillary acidic protein) (BD Bioscience).
  • Notch-1 and Nestin positive cells were identified by immune-histochemistry in dental follicle slices ( FIG. 1 . ⁇ and ⁇ ), which were described as markers of putative stem cells in dental epithelium and neural stem cells (Harada et al. 1999, Johansson et al. 1999b), but were negative for CD133, VEGFR-2 and GFAP (table B).
  • FIG. 1 shows immunephenotype of dental follicle. Studies based on immunoperoxidase reactivity were performed with slices of dental follicle. Antibodies: ⁇ ) Notch-,1 ⁇ ) Nestin.
  • osteoprogenitors can be isolated from aspirates of bone marrow by their ability to adhere to a plastic substratum or colony forming unit fibroblast (CFU-F).
  • CFU-F colony forming unit fibroblast
  • FIG. 2 ⁇ The presence of a clonogenic and plastic adherent cell population in dental follicle tissue ( FIG. 2 ⁇ ). is demonstrated.
  • the cells within each colony were characterized by a typical fibroblast-like morphology analogous to the progeny of human bone marrow CFU-F and they were positive for Notch-1 and Nestin by immunochemistry (FIGS. 2 ⁇ and ⁇ ).
  • FIG. 2 shows ⁇ ) Representative colony after 14 days. ⁇ ) and ⁇ ) characterize immunophenotypes of cultured DFSC with representative staining pattern for ⁇ ) Nestin and ⁇ ) Notch-1.
  • DPSC and BMSC express collagen1, bonesialoprotein, osteocalcin and phex. These genes are typically for osteoprogenitor cells. Both cell types express FGFR1-IIIC, which were detected also by fibroblast isolated from the periodontal ligament, but not CD34 and CD133, common marker for haematopoietic stem cells. We detected faint differences for IGF-2 and FGFR3-IIIC for gene expression. Interestingly The intensity of the IGF-2 signal was better for DFSC than for BMSC. Because of the missing differences DFSC and BMSC were investigated with qPCR (FIG. C). It was detected that-IGF-2 expression in DFSC cells was more than 100 time increased compared to the BMSC cells, in contrast the expression of BS and OCN was decreased (15 times).
  • follicle cells as cementoblast progenitor cells which were unable to differentiate into mineralised cells in vitro, but in vivo (Handa K. et al. (2002) bone 31(5): 606-611). DFSCs maturated probably in knock-out D-MEM serumreplacement media to spheroid-like structures.
  • FIG. 3 a shows QPCR with cultured BMSC and DFSC grown in MSCBM BulletKit. Values for relative concentration were calculated with delta/delta method (Winer et al. 1999), total RNA from BMSCs were used as calibrator. House-keeping gene was GAPDH.
  • FIG. 3 b shows QPCR with cultured DFSC grown in MSCBM BulletKit, D-MEM supplemented with 20% FCS and knockout D-MEM (serumreplacement). Values were calibrated with total RNA derived from DFSC cultured in MSCBM Bulletkit.
  • Clones were examined using insulin and dexamethasone to determine their identity, i.e., either lineage-committed progenitor cells or lineage-uncommitted pluripotent cells.
  • Uineage-induction agents such as dexamethasone, induce lineage-commitment and expression in pluripotent cells, but does not alter phenotypic expression in progenitor cells.
  • Cultures treated with dexamethasone will exhibit multiple expressed phenotypes. Thus comparing the effects of treatment with dexamethasone and insulin can identify specific types of progenitor and pluripotent cells within an unknown group of cells.
  • BMSC culture produced extensive sheets of calcified deposits over the entire adherent layer after 6 weeks of induction ( FIG. 4 ).
  • FIG. 4 shows the differentiation of DFSC and BMSC in vitro: Adherent layers of cultured DFSCs ( ⁇ , ⁇ ) and BMSC ( ⁇ ), are shown with Alizarin Red staining as a measure of calcium accumulation following 5 ( ⁇ ) and 6 weeks ( ⁇ , ⁇ ) of induction with DMEM with 10% FCS, L-acorbate-2-phosphate, inorganic phosphate and dexamethasone.
  • FIG. 5 shows differentiation of DFSC to a novel follicle-tissue-membrane (FTM) in vitro: Culture bottle with layer of cultured DFSCs are shown with Alizarin Red staining as a measure of calcium accumulation following 5 weeks of induction with DMEM with 10% FCS, L-acorbate-2-phosphate, inorganic phosphate and dexamethasone.
  • FTM follicle-tissue-membrane
  • FIG. 6 shows QPCR with cDNA from DFSCs cultured in media with and without dexamethason: For abbreviation see above.
  • FIG. 7 shows HE- and trichrom-stainings with FTM-slices.
  • Biodegradable Polymers as scaffolds for engineering of teeth, and teeth related tissues such as nerves, bones and connecting tissue (periodontium).
  • compositions of the present invention may comprise the pluripotent embryonic-like stem cells of the present invention, or cells, tissues or organs derived there from, alone or in a polymeric carrier or extracellular matrix.
  • Suitable polymeric carriers include porous meshes or sponges formed of synthetic or natural polymers, as well as polymer solutions.
  • One form of matrix is a polymeric mesh or sponge; the other is a polymeric hydrogel.
  • Natural polymers that can be used include proteins such as collagen, albumin, and fibrin; and polysaccharides such as alginate and polymers of hyaluronic acid. Synthetic polymers include both biodegradable and non-biodegradable polymers.
  • Biodegradable polymers are applicable to those tissue engineering products for which tissue repair or remodelling is the goal, but not where long-term materials stability is required. Biodegradable polymers must also possess
  • biodegradable polymers include polymers of hydroxy acids such as polylactic acid (PLA), polglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA), poly-orthoesters, polyanhydrides, polyphosphazenes, and combinations thereof.
  • PLA polylactic acid
  • PGA polglycolic acid
  • PLGA polylactic acid-glycolic acid
  • poly-orthoesters polyanhydrides
  • polyphosphazenes polyphosphazenes
  • Collagen is one of the most important scoffold for tissue engineering.
  • Glycosaminoglycans which consist of repeating disaccharide units in linear arrangement, usually include an uronic acid component (such as glucuronic acid) and a hexosamine component (such as n-acetyl-d-glucosamine).
  • uronic acid component such as glucuronic acid
  • hexosamine component such as n-acetyl-d-glucosamine.
  • the predominant types of GAGs attached to naturally occurring core proteins of proeoglycans include chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate.
  • the GAGs are attached to the core protein by specific carbohydrate sequences containing three or four monosaccharides.
  • Hyaluronic acid is an anionic polysaccharide with repeating disaccharide units of n-acetylglucosamine and glucoronic acid, with unbrached units ranging from 500 to several thousand.
  • Hyaluronic acid can be isolated from natural sources (e.g., rooster combs) or via microbial fermentation. Because of its water-binding capacity, dilute solutions of hyaluronic acid are viscous.
  • hyaluronic acid can easily chemically modified, as by etherification of the carboxyl moieties, which reduces its water solubility and increases its viscosity
  • Hyaluronic acid can be cross-linked to form molecular weight complexes in the range 8 to 24 ⁇ 10 6 or to form an infinite molecular network (gels).
  • hyaluronic acid is cross-linked using aldehydes and small proteins to form bonds between the C—OH groups of the polysaccharide and the amino to imino groups of the protein, thus yielding high-molecular-weight complexes.
  • the resultant infinite-network gels can be formed into sheaths, membranes, tubes, sleeves, and particles of various shapes and sizes.
  • PHA polyesters are degradable, biocompatible, thermoplastic materials made by several microorganisms. They are intracellular storage polymers whose function is to provide a reserve of carbon and energy. PHB and it copolymers with up to 30% of 3-hydroxyvaleric acid are now commercially available under the trade name Biopol.
  • Degradable tyrosine-polycarbonates are an other important polymer for cell adhesion and tissue engineering.
  • Naturally occurring hydroxy acids such as glycolic, lactic, and E-caproic acids
  • hydroxy acids such as glycolic, lactic, and E-caproic acids
  • bioresorbable surgical sutures made from poly( ⁇ -hydroxy acids) have been in clinical use since 1970; other implantable devices made from these versatile polymers (e.g. internal fixation devices for orthopedic repair) are becoming part of standard surgical protocol.
  • poly(hydroxy acid) implants involve inflammatory response can have a deleterious effect on some healing events, these polymers have been successfully employed as matrices for cell transplantation and tissue regeneration.
  • Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohols. Polymers that can form ionic or covalently crosslinked hydrogels which are malleable—are used to encapsulate cells.
  • a hydrogel is a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a get.
  • Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block copolymers such as Pluronics' or Tetronics', polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively.
  • these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charge a side groups, or a monovalent ionic salt thereof.
  • Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.
  • acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.
  • polymers with basic side groups that can be reacted with anions are poly(vinyl amines), poly(vinyl pyridine), poly(vinyl imidazole), and some imino substituted polyphosphazenes.
  • the ammonium or quaternary salt of the polymers can also be formed from the backbone nitrogens or pendant imino groups.
  • basic side groups are amino and imino groups.
  • the solvent casting of PLLA is used with dispersed NaCl particles, which can be leached out by water forming interconnecting pores of 50-300 ⁇ m.
  • Periodontitis is a disease that results in the destruction of supporting tissues of the teeth, subsequent gingival pocket formation, and ultimately tooth loss.
  • the main etiology is bacterial infection, but occlusal and systemic disorders are considered aggravating factors.
  • periodontitis is not life threatening, it is one of the most widespread disorders found among human populations.
  • Most surveys in Europe state that more than 40% of persons in the age group of 35-45 years old present periodontal pocketing. The periodontal status deteriorates as the subjects grow older: In France the percentage of people with periodontal exceeds 60% in the age group 45-64 years old. In the United States 36% of the population 19 years and older presents periodontitis; this increases to 52.2% of persons 45-64 years old).
  • Health costs for this disease have been estimated at $ 5-6 billion involving 120-133 million hours of treatment, even though most patients do not seek treatment.
  • the gingival can bind only to the vascular surface of the teeth, and very often the adherence is too frail, or the blood clot is resorbed too rapidly. In this case, a long junctional epithelium may develop, between the tooth and the flap, resulting in the formation of a pocket. An additional complication is the accumulation of plaque. Oral microorganism adhere to the portion of the tooth surface that is next to the marginal gingival. Because of these wound closure hindrances, complete submersion of the teeth under gingival flaps has been advocated but in seldom feasible in human clinical situations.
  • New advances in periodontal therapy include the use of membrane barriers to guide the regenerative tissue, and implantation of bone-substitutes. These techniques apply to vertical bone lesions and are becoming quite efficacious and predictable. Promoting regeneration at a distance from a potential source of osseous cell remains difficult, but better knowledge of cheramoattractants, growth factors, and osteoinduction proteins is likely to help to develop clinical applications that enhance healing and facilitate the selection of proper cell populations for bone or gingival reconstruction.
  • the introduction of teeth derived stem cells for vascularization, regeneration periodontal tissue, gingival tissue and bone is an important step for improvement of guided tissue regeneration.
  • Tissue Regeneration is an important field for regeneration processes improved by stem cells, and s.c. derived or stimulated cell lines.
  • the presence of gingival is detrimental to the regeneration of the attachment apparatus.
  • Nyman and co-workers were the first to find an effective way of excluding gingival epithelial and connective tissue cells from reconstruction sites.
  • Their initial attempt to regenerate periodontium in the human succeeded in forming 5 mm of new attachment with collagen fibers embedded in cementum. They used a Millipore filter, which served as a barrier to keep connective tissue cells out of the resorbed site and avoid downward growth of the gingival epithelium along the root surface. Little bone was produced, perhaps because the filter collapsed against the tooth.
  • e-PTFE expanded poly(tetrafluoroethylene)
  • Teflon expanded poly(tetrafluoroethylene)
  • the advantages of e-PTFE are its stiffness, that it allows more room for bone formation, is biocompatible, and the ease with which it can be removed. Numerous studies have proved the ability of e-PTFE membranes to promote repair of intrabony and furcation defects. Histologic confirmation of the repair, evidencing neoformation of cementum, periodontal ligament, and alveolar bone, was obtained in both humans and animals).
  • the material has, however, two major drawbacks: it causes severe gingival recession and, once exposed, accumulates large amounts of bacterial plaque. Vascularization of the marginal gingival is provided solely by terminal capillaries.
  • a cell-tight barrier disturbs the blood supply and avoids endothelial anastomosis with blood vessels originating from the underlying tissues. Bacteria actively proliferating in the crevicular part of the tooth will then colonize the e-PTFE membrane. Because of the subsequent inflammation, the Teflon membrane must be removed between 4 and 6 weeks after replacement).
  • resorbable membranes have been developed using collagen or polysaccharide polymers. Collagen membranes used experimentally can be made from rat-tail, bovine dermis, or other sources. Industrially produced collagen membranes include collagen from calf endocardium and are in use in human clinics.
  • poly(lactic acid), poly(galactic acid) or a novel product dissolves lactic acid in N-methyl-2-pyrolidone have been used to form polymer membranes.
  • resorbable membranes When exposed, resorbable membranes accumulate small amounts of bacterial plaque because bacterial and salivary enzymes accelerate their resorption. Thus permeabilized, the membrane is partially protected by the tissue exudates.
  • Many polymer barriers feature pores that allow fluid circulation or even alimited amount of tissue ingrowth. The resorption rate of resorbable barriers can modulated and thus their effect prolonged if needed. How long a membrane should stay in place has not been determined.
  • Periodontal ligament cells migrated several millimetres along the implant, producing cementum and a periodontal-like ligament. The cells were able to colonize a fairly large surface much faster than the osseous cells crossed the minute space left by the drill. It seems then that these two types of cells do not compete, but primarily restore their intended functions.
  • membranes should be positioned under the flap margin.
  • barrier membranes promotes predictable osseous repair and histologically verifiable new attachment with neoformation of cementum and periodontal ligament fibers.
  • transplantation of Stem Cells derived from follicle cells can enforce remodelling of periodontium.
  • Intraoral autologous bone grafts are, of course, choice material when osseous defects need to be repaired.
  • intraoral autologous bone is not always available in sufficient quantities, particularly when patients present a deficiency of alveolar bone resulting from severe periodontitis.
  • Bone substitutes include synthetic materials such as hydroxyapatite and methyl methacrylate, and modified mineralised biologic substances such as coralline or bovine trabecular bone and lyophilised human bone. These products provide surgical ease and patient comfort, and their various resorption rates are interesting. Opponents of bone grafting techniques affirm that regeneration of the periodontal attachment apparatus is not achievable by, or at least has not been evidenced for, procedures other than membranes implantations). The best results were obtained when teeth were submerged under flaps or when the sites were protected from epithelial downward growth by gingival grafts). Bone substitutes have been suspected to impair the regeneration of connective tissue attachment.
  • Fibronectin has been considered a promising promoter of fibroblastic activity, and that topical application of fibronectin on demineralized roots could be beneficial to healing.
  • fibroblast growth factor FGF
  • FGF fibroblast growth factor
  • FGF has a potent chemotactic effect on fibrobalsts derived from periodontal ligament tissues. FGF is 10,000 time more powerful than fibronectin in inducing fibroblast growth.
  • Platelet-derived growth factor is another chemoattractant with great potential, and no amount of fibronectin can stimulate the proliferation of periodontal ligament fibroblasts to the extent achieved by nanograms of PDGF
  • the usefulness of fibronectin can also be questioned, because it has been shown that it enhances the migration of basal keratinocytes (Where epithelial cells colonize the root before connective tissue is formed, there will be no attachment apparatus. Therefore, although fibronectin attracts and orients fibroblasts properly, some of its effects might not be desirable.
  • Biologic glues have also been proposed as spacers to keep membrane barriers at a distance from the roots, but the usefulness of fibrinogen and/or fibronectin in periodontal surgery is still inconclusive—It is possible to put stem cells into biological glue.
  • FGF and PDGF both have powerful effects on periodontal ligament fibroblasts.
  • Periodontists have also shown interest in transforming growth factor ⁇ (TGF- ⁇ ; which appears to promote more significant deposition of fibroblast collagen than any of the other growth factors, although it is not a fibroblast chemoattractant.
  • TGF- ⁇ transforming growth factor ⁇
  • IGF-1 insulin-like growth factor
  • the PDGF-IGF-1 combination is chemotactic and mitogenic for periodontal fibroblasts; PDGF is chemotactic for osteoblasts, and promotes the synthesis of noncollageneous proteins in bone cultures, and IGF-1 promotes the synthesis of collagen proteins.
  • the PDGF-IGF-1 combination results in greater bone matrix formation than any individual growth factor, and interacts synergistically to yield significant collagen formation and healing in soft tissue wounds).
  • the delivery of growth factors through a gel allows the presence of a great amount of PDGF and IGF-1 during a short period of time, and even better results could perhaps be obtained with a slow-release system. Repair requires several weeks and it will be interesting to develop acceleration with Stem Cells slow-release devices.
  • Bone morphogenic proteins (BMPs) a mixture of peptides initially identified as a bone growth factor and later shown to comprise at least 13 individual proteins can promote new bone formation at the site of implantation, instead of changing the bone growth rate of existing bone.
  • RhBMP2 human bone morphogenic protein 2
  • RhBMP2 recombinant human bone morphogenic protein 2
  • RhBMP in a carrier enhances periodontal regeneration.
  • the membrane favors migration and proliferation of already RhBMP promotes the grouping of mesenchymal stem cells in clusters, later developing in appropriate tissues.
  • RhBMP may promote vertical bone formation in horizontal osseous defects. The abundant bone produced not only covered the denuded half-root but some cases even grew over portions of the crowns of the teeth.
  • PDL-CTX A small 12.5-kDa peptide, PDL-CTX, also seems to be a potent chemoattractant for periodontal ligament cells, with mitogenic properties or cells in early stages such as stem cells.
  • the growth factor amelogenin found in the epithelial cells forming the enamel organ, has been extracted from pigs and results in neoformation of a acellular cementum. Amelogenin increases proliferation of ligament fibroblasts but not of epithelial cells.
  • Amelegenin is a potent promotor for stem cell derived tissue engineering.
  • Dentin Powder may be used as dentinogenial transmitter substance for seeding stem cells.
  • Crude dentin fractions prepared by ethylenediaminetetraacetic acid (EDTA) extraction and collagerase digestion stimulate reparative dentinogenesis in small pulp exposures in ferrets or reactive dentinogenesis when applied to freshly cut dentin surfaces.
  • Some extracellular matrix component is a prerequisite to odontoblast differentiation and tubular dentin formation.
  • To regulate odontoblast differentiation and hence the architecture of the tissue may possible by stem cells implantation. Regulation of dentin architecture may be important.
  • Clusters of blood vessels are found ramifying around the tooth germ in the dental follicle and entering the dental papilla (or pulp) during the cap stage. Their number in the papilla increases during histodifferentiation, reaching a maximum at the onset of the crown stage of tooth development. Interestingly, the vessels entering the papilla are clustered into groups that coincide with the position where the roots will form. With age, the volume of pulpal tissue diminishes and the blood supply becomes progressively reduced, affecting the tissue's viability.
  • the dental organ derived solely from epithelium, is avascular, although a heavy concentration of vessels in the follicle exists adjacent to the outer dental epithelium.
  • Biodegradable scaffolds in the shape of human molars, seeded them with stem cells derived from stem cells cultured in D-MEM serumreplacement media (GIBCO/BRL) supplemented with 2 mM L-glutamine/100 units/ml penicillin/100 ⁇ g/ml streptomycin (Biofldids, Rockville, Md.), and implanted the cell/polymer constructs in a cell rotation reactor type from Syntecon, Rotary Cell Culture System RCCS-D, with 50 ml vessel (D-405).
  • FIG. 8 shows a fixed section of scaffold with cultured stem cells (stained with Hoechst 33258).
  • FIG. 9 shows pore structure of PLLA-polymer (80 ⁇ magnification) TABLE 1 PLLA cast over non woven PGA fibers PGA - mesh by PLLA PLLA microstructures PLGA blending with PEG PLLA soaking in PVA Printing: 3-D Printing Emulsion - freeze drying process Polymer-ceramic: Hydroxyapatite in PLGA - PVA Hydroxyapatite PLGA Polyhydroxybutyrat and copolymers Biopol Polydioxanone Poly ( ⁇ ) - caprolactone Polyorthoesters - polythyrosine-carbonates Polyanhydrides (F-K-Gliadel) Polyphosphazene Polyaminoacids Polyaminocarbonat - polyaminoocarbonat-tyrosine Photopolymers Mixtures thereof.
  • a matrix having cadherins enhancing the binding of heterophilics which are lectins-like e.g. branched Oligosaccharide structures namely the sialyl X and A structures.
  • a matrix having the immunglobuline family proteins that are denoted as cell adhesion molecules (CAMs) for homophilic and heterophilic binding.
  • CAMs cell adhesion molecules
  • a matrix having integrins, dimeric molecules, consisting of the ⁇ and ⁇ subunit for cell-cell and for cell-matrix-binding (collagen, fibronectin, vitronectin, vWF, and lamin).
  • A shows the tooth in situ.
  • B is filled with clot.
  • the clot resolves, C, by (1) the polymorph response, (2) the macrophage response, and (3) the fibroblast response.
  • the bohy defect becomes colonized by new osteoblasts (4) that remodel the collagen scar and form bone, D.
  • Implantation of stem cells derived from teeth are able to regenerate periodontium in any stage of healing (a-d) with and without a guiding scaffold (Membrane).
  • the epithelial response is achieved by proliferation and migration of cells to cover the defect.
  • the connective tissue response involves successively (1) a polymorph response (12 to 24 hours), (2) a macrophage response (2 to 5 days), and (3) a fibroblast response (2 days and on) from undifferentiated perivascular cells and undamaged fibroblasts.
  • the new fibroblasts (4) form the collagen of scar tissue.
  • stem cells from teeth into the wound bed For improvement of wound healing, it is possible to introduce stem cells from teeth into the wound bed.
  • the repair process up to step 4 is as depicted in FIG. 18-7 involving (1) the polymorph response, (2) the macrophage response, (3) fibroblast differentiation, and (4) collagen synthesis.
  • the scar remodels (5) by collagen phagocytosis.

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US20070254005A1 (en) * 2004-08-26 2007-11-01 Pathak Chandraskekhar P Implantable Tissue Compositions and Method
US20070258957A1 (en) * 2006-05-04 2007-11-08 Russell Bowermaster Method for obtaining and storing multipotent stem cells
US20100172863A1 (en) * 2009-01-03 2010-07-08 Wasielewski Ray C Enhanced medical implant
US20100285582A1 (en) * 2007-10-01 2010-11-11 Seoul National University Industry Foundation Various human dental stem cells having a mineralization ability and the method for culturing them
US20110158962A1 (en) * 2008-06-26 2011-06-30 Federico Ferro Dental pulp marrow similar cells (dpmsc) and methods of isolating and using
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US20130243739A1 (en) * 2012-03-19 2013-09-19 Richard Burt Methods And Compositions For Regenerating And Repairing Damaged Or Aged Tissue Or Organs Using Nonviable Irradiated Or Lyophilized Pluripotent Stem Cells
US20140128596A1 (en) * 2009-03-03 2014-05-08 Access Medical Systems, Ltd. Crosslinked polysaccharide
US9867855B2 (en) 2006-07-20 2018-01-16 Richard Burt Method of using mitotically inactivated stem cells for damaged tissue repair
US10328103B2 (en) 2009-01-03 2019-06-25 Ray C. Wasielewski Medical treatment composition comprising mammalian dental pulp stem cells
US10813955B2 (en) 2015-09-29 2020-10-27 Genani Corporation Methods for treating age-related organ or tissue dysfunction through heterochronic transbiosis using nonviable pluripotent stem cells

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US20040058442A1 (en) * 2000-07-21 2004-03-25 Songtao Shi Adult human dental pulp stem cells in vitro and in vivo
US20110177150A1 (en) * 2004-08-26 2011-07-21 Pathak Holdings, Llc Implantable tissue compositions and method
US20070254005A1 (en) * 2004-08-26 2007-11-01 Pathak Chandraskekhar P Implantable Tissue Compositions and Method
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US20070134624A1 (en) * 2005-12-09 2007-06-14 Krasner Paul R Apparatus and method for storing and transporting teeth
US7448871B2 (en) 2005-12-09 2008-11-11 Krasner Paul R Apparatus and method for storing and transporting teeth
US20090130753A1 (en) * 2006-05-04 2009-05-21 Russell Bowermaster Method for Obtaining and Storing Multipotent Stem Cells
US20080176325A1 (en) * 2006-05-04 2008-07-24 Russell Bowermaster Method for Obtaining and Storing Multipotent Stem Cells
US20070258957A1 (en) * 2006-05-04 2007-11-08 Russell Bowermaster Method for obtaining and storing multipotent stem cells
US9867855B2 (en) 2006-07-20 2018-01-16 Richard Burt Method of using mitotically inactivated stem cells for damaged tissue repair
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US20100285582A1 (en) * 2007-10-01 2010-11-11 Seoul National University Industry Foundation Various human dental stem cells having a mineralization ability and the method for culturing them
US10421947B2 (en) 2008-06-26 2019-09-24 Foundation For Translational Science Dental pulp marrow similar cells (DPMSC) and methods of isolating and using
US20110158962A1 (en) * 2008-06-26 2011-06-30 Federico Ferro Dental pulp marrow similar cells (dpmsc) and methods of isolating and using
US8470308B2 (en) 2009-01-03 2013-06-25 Ray C. Wasielewski Enhanced medical implant comprising disrupted tooth pulp and tooth particles
US20100209878A1 (en) * 2009-01-03 2010-08-19 Wasielewski Ray C Enhanced medical implant
US20100209387A1 (en) * 2009-01-03 2010-08-19 Wasielewski Ray C Enhanced medical implant
US8470309B2 (en) 2009-01-03 2013-06-25 Ray C. Wasielewski Enhanced medical implant comprising disrupted tooth pulp and tooth particles
US20100172863A1 (en) * 2009-01-03 2010-07-08 Wasielewski Ray C Enhanced medical implant
US8540978B2 (en) 2009-01-03 2013-09-24 Ray C. Wasielewski Treatment composition comprising physically disrupted tooth pulp and non-cultured stem cells in a matrix
US8562969B2 (en) 2009-01-03 2013-10-22 Ray C. Wasielewski Treatment composition comprising physically disrupted tooth pulp and non-cultured stem cells
US10335436B2 (en) 2009-01-03 2019-07-02 Ray C. Wasielewski Medical treatment composition comprising mammalian dental pulp stem cells
US10328103B2 (en) 2009-01-03 2019-06-25 Ray C. Wasielewski Medical treatment composition comprising mammalian dental pulp stem cells
US20100215617A1 (en) * 2009-01-03 2010-08-26 Wasielewski Ray C Enhanced medical implant
US9434789B2 (en) * 2009-03-03 2016-09-06 Access Medical Systems, Ltd. Crosslinked polysaccharide
JP2015166750A (ja) * 2009-03-03 2015-09-24 アクセス メディカル システムズ，リミティド 高感度蛍光分析のための検出システム及び方法
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US9381151B2 (en) 2012-03-19 2016-07-05 Richard Burt Methods and compositions for regenerating and repairing damaged or aged tissue or organs using nonviable irradiated or lyophilized pluripotent stem cells
US9757422B2 (en) 2012-03-19 2017-09-12 Richard K. Burt Methods and compositions for regenerating and repairing damaged or aged tissue or organs using nonviable irradiated or lyophilized pluripotent stem cells
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