EP3452500A1 - Mesenchymal stem cell proliferation - Google Patents
Mesenchymal stem cell proliferationInfo
- Publication number
- EP3452500A1 EP3452500A1 EP17793552.5A EP17793552A EP3452500A1 EP 3452500 A1 EP3452500 A1 EP 3452500A1 EP 17793552 A EP17793552 A EP 17793552A EP 3452500 A1 EP3452500 A1 EP 3452500A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sprf
- mscs
- patient
- vivo
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- 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
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3616—Blood, e.g. platelet-rich plasma
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
- C12N2502/115—Platelets, megakaryocytes
Definitions
- the invention relates to a new method for increasing proliferation rate of mesenchymal stem cells.
- MSCs Mesenchymal stem cells
- ALCAM activated leukocyte cell adhesion molecule, CD166
- STRO-1 surface markers
- Their multipotency permits the differentiation to bone, cartilage, reticular tissues and fat (Oreffo et al. 2006). Due to their advantageous properties MSCs have been proved to be effective as autologous cell transplantation in clinical trials in case of regeneration of periodontal tissue defects, diabetic critical limb ischemia, bone damage caused by osteonecrosis and burn-induced skin defects.
- MSCs multi-lineage potential can be lost easily, when MSCs grown in vitro on standard tissue culture plastics. Their proliferation and multilineage differentiation potential also decreases with aging or increased time in in vitro culture.
- MSCs fetal bovine serum
- FBS fetal bovine serum
- platelet-rich plasma was already proven in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration.
- Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared.
- Amable P. R. et al. Stem Cell Research & Therapy2013, 4:67, Rigotti G. et al. Aesthet Surg J. 2016; 36(3):261-70 2013 suggested a standardized PRP and the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes. Uncertainties of the process are involved.
- Plasma is the anticoagulated, centrifuged whole blood supernatant, which has the disadvantage of containing anticoagulant (e.g. EDTA, heparin or citrate derivatives), which affects enzymatic balance and interfere with systemic blood coagulation as well and plasma also contains fibrinogen, which is converted to fibrin just like in PRP and causes limited protein transport.
- anticoagulant e.g. EDTA, heparin or citrate derivatives
- fibrinogen which is converted to fibrin just like in PRP and causes limited protein transport.
- Another possible candidate is serum, which is the supernatant of the coagulated whole blood.
- ACS autologous conditioned serum
- the main criteria are to have a somewhat standardized, fibrin and/or fibrinogen and anticoagulant free autologous blood separation product, which does not induce inflammation.
- the only procedure, which enabled to fulfil these goals was simply centrifuging whole blood after blood drawing in a clot activating tube so platelet rich fibrin (PRF) is produced in the supernatant. Pressing out the fluid content from PRF leads to an autologous blood separation product, which does not contain fibrinogen, anticoagulants and the inflammation markers are low.
- PRF platelet rich fibrin
- SPRF platelet rich fibrin
- DMEM Dulbecco' s modified Eagle' s medium
- FBS fetal bovine serum
- hMSCs human mesenchymal stem cells
- hSBPs human subchondral bone pieces
- SPRF serum from platelet rich fibrin
- the invention relates to a method for use of serum fraction of platelet rich fibrin (SPRF) for increasing mesenchymal stem cell (MSC) proliferation rate in vitro, ex vivo or in vivo wherein said MSCs maintain their potential to differentiate into several cell types.
- SPRF platelet rich fibrin
- the invention relates to a use of serum fraction of platelet rich fibrin (SPRF) for increasing MSC proliferation rate in vitro, ex vivo or in vivo wherein said MSCs maintain their potential to differentiate into several cell types.
- SPRF platelet rich fibrin
- the MSCs are contacted or maintained in contact with
- SPRF for at least 5 days, preferably for at least 8 days or at least 10 days.
- the MSCs are contacted or maintained in contact with SPRF for until at least a time -period when osteoblast direction differentiation occurs, preferably for until at least a time-period when the expression of at least one, preferably two or at least two osteoblast specific marker gene(s) is/are increased in a medium supplemented with SPRF, preferably SPRF having the concentration range given herein, highly preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium.
- osteogenic marker genes Preferably expression of one or both of the following osteogenic marker genes is/are increased:
- - COL1A1 expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold,
- - ALPL expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold, when compared to 10 % (v/v) FCS supplemented medium.
- MSC culturing medi- um normally comprises a carbon source eg. sugar source eg. glucose.
- the medium comprises SPRF and comprises no further serum and/or no serum substitute and/or no serum derived product or supplement.
- the medium according to the invention does not comprise fetal bovine (calf) serum (FBS or FCS) and does not comprise platelet rich plasma (PRP).
- the medium according to the invention does not comprise any further growth factor (only those which are present in the SPRF).
- MSCs are obtained from a subject, said method comprising
- the MSCs so proliferated are administered to a subject.
- the subject is a patient in need of bone or cartilage regeneration or repair.
- the patient is treated with a condition wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.
- the patient is in need of bone tissue regeneration.
- the patient is suffering in osteoarthritis or osteoarthritis, preferably osteoarthritis or osteoarthritis of a joint.
- the patient is a mammalian or human subject.
- the MSCs are present in or on a tissue and so cultured or maintained in culture.
- the tissue is an explant. In an embodiment the tissue is an artificial tissue. In an embodiment the tissue is an explant, eg. a bone explant. The bone explants may be e.g. subchondral bone pieces or explants obtained by osteotomy. In an embodiment the tissue is an artificial tissue, e.g. a bone graft or a joint or cartilage graft.
- an MSC culturing medium for maintaining or culturing a tissue ex vivo is a medium for culturing mammalian cells e.g. a medium for culturing MSCs supplemented with SPRF.
- MSC culturing medium normally comprises a carbon source eg. sugar source eg. glucose, a glutamine source and pyruvate.
- the medium comprises SPRF and no further serum and/or no serum substitute and/or no serum derived product or supplement.
- the medium according to the invention does not comprise fetal bovine (calf) serum (FBS or FCS) and does not comprise platelet rich plasma (PRP).
- the medium according to the invention does not comprise any further growth factor (only those which are present in the SPRF).
- MSCs are obtained from a subject, said method comprising
- the MSCs are incubated in the presence of SPRF for at least 5 days, preferably for at least 8 days or at least 10 days.
- the MSCs are bone marrow derived mesenchymal stem cells (BM-MSCs or bone mar- row stromal stem cells).
- BM-MSCs bone marrow derived mesenchymal stem cells
- bone mar- row stromal stem cells bone mar- row stromal stem cells
- the MSCs are adipose derived mesenchymal stem cells (AD-MSC).
- AD-MSC adipose derived mesenchymal stem cells
- the medium is as defined above or a medium as disclosed herein.
- tissue or explant on which MSCs so proliferated is/are administered to a subject.
- tissue or explant is a graft to be implanted into the subject.
- the subject is a subject in need of bone or cartilage regeneration or repair.
- the subject is suffering in osteoarthritis or osteoarthritis, preferably osteoarthritis or osteoarthritis of a joint.
- the subject is a mammalian or human subject.
- the MSCs are mammalian MSCs, preferably human MSCs.
- the invention relates to a method for use of serum fraction of platelet rich fibrin (SPRF) for increasing mesenchymal stem cell (MSC) proliferation rate in vitro wherein said MSCs maintain their potential to differentiate into several cell types.
- SPRF platelet rich fibrin
- the invention relates to a use of serum fraction of platelet rich fibrin (SPRF) for increasing MSC prolif- eration rate in vitro wherein said MSCs maintain their potential to differentiate into several cell types.
- SPRF platelet rich fibrin
- the expression of at least one or two, preferably two or at least two osteoblast differentiation factors show increased expression after an appropriate period of time, preferably at or after 5 days culturing.
- the osteoblast factors are COL1 Al and ALPL.
- the MSCs are incubated in the presence of SPRF for at least 5 days, preferably for at least 8 days or at least 10 days.
- the MSCs are obtained from a subject.
- the subject is a mammalian subject, more preferably a human subject.
- the MSCs are primary cells.
- the MSCs are bone marrow derived mesenchymal stem cells (BM-MSCs or bone mar- row stromal stem cells).
- BM-MSCs bone marrow derived mesenchymal stem cells
- bone mar- row stromal stem cells bone mar- row stromal stem cells
- the MSCs are adipose derived mesenchymal stem cells (AD-MSC).
- AD-MSC adipose derived mesenchymal stem cells
- the medium is as defined above or a medium as disclosed herein.
- the culture of mesenchymal stem cells is supplemented with 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.
- one or more amino acid source preferably at least glutamate source or a glutamate source only, - one or more salts, said salt being preferably selected from calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and monosodium phosphate,
- one or more sugar preferably at least glucose or glucose only and optionally or if desired one or more vitamins preferably selected from folic acid, nicotinamide, riboflavin and B12, wherein said medium comprises 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF wherein said medium comprises no FBS (FCS) and no PRP and preferably no FGF, wherein preferably said medium comprises, besides SPRF, no other serum product and/or no other serum derived product or supplement and preferably no other growth factors.
- FCS FBS
- PRP preferably no FGF
- the medium may comprise further additives e.g. buffer(s), antibiotic(s), selection agent(s), preservation agent(s) etc.
- the medium is a derivative of Dulbecco' s modified Eagle's medium (DMEM) in that it is supplemented with 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.
- DMEM Dulbecco' s modified Eagle's medium
- SPRF is contacted with MSCs of a subject in vivo and
- SPRF mesenchymal stem cell
- no other serum derived product or supplement and preferably no other growth factors are administered to the subject besides SPRF.
- the MSCs are maintained in contact with or in the pres- ence of SPRF in vivo for at least 5 days, preferably for at least 8 days or at least 10 days.
- the subject is in need of regeneration of cartilage and/or bone
- SPRF is administered to a site wherein it may be contacted with the bone or cartilage to be regenerated
- MSCs present at the site of administration are contacted or maintained in contact with SPRF, and
- SPRF level is maintained to proliferate MSCs for until at least a time -period when osteoblast direction differentiation occurs, preferably for until at least a time -period when the expression of at least one, preferably two or at least two osteoblast specific marker gene(s) is/are increased in a medium supplemented with SPRF, preferably SPRF having the concentration range given herein, highly preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium.
- the MSCs present at the site of administration in the subject are MSCs propagated according to the present invention, preferably MSCs obtained from a subject, cultured and propagated in vitro and reintroduced or readministrated to said subject.
- osteogenic marker gene/s Preferably expression of one or both of the following osteogenic marker gene/s is/are increased:
- - COL1A1 expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold,
- the subject is a mammalian subject, more preferably a human subject.
- the MSCs are bone marrow derived mesenchymal stem cells (BM-MSCs or bone marrow stromal stem cells).
- BM-MSCs bone marrow derived mesenchymal stem cells
- bone marrow stromal stem cells bone marrow derived mesenchymal stem cells
- the MSCs are adipose derived mesenchymal stem cells (AD-MSC).
- AD-MSC adipose derived mesenchymal stem cells
- the expression level of the following MSC-specific genes is unchanged or is increased by 1 to 150% in comparison with the same medium supplemented with the same concentration of FCS instead of SPRF or in comparison with the same medi- um supplemented with 10% of FCS instead of SPRF.
- the expression level is measured by real time quantitative PCR (rt-qPCR).
- the expression level is measured on or after 5 days as of starting the administration of SPRF or contacting the cells with SPRF.
- the expression levels of the following MSC marker genes are increased: ALCAM (CD166), ITGB l, CD105, ANPEP.
- the expression of hMSC-specific genes are increased after 5 days incubation in a medium supplemented with SPRF, preferably SPRF having the concentration range given above, preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium as follows:
- - ALCAM expression is increased at least 1.2 fold (by 20%), preferably at least 1.4 fold or 1.6 fold, - ITGBl expression is increased at least 1.5 fold (by 50%), preferably at least 1.7 fold or 1.9 fold,
- - CD105 expression is increased at least 1.05 fold (by 5%), preferably at least 1.1 fold or 1.2 fold and
- - ANPEP expression is increased at least 1.1 fold (by 10%), preferably at least 1.2 fold or 1.3 fold, preferably as confirmed by real time qPCR.
- any one of the above markers is at least not decreased.
- no adipose differentiation occurs in the MSCs when MSCs are cultured in SPRF, preferably
- the expression level of adipogenic (adipocyte) markers FABP4, PPARG and ADIPOQ expression, that are markers of adipogenic differentiation is not increased by more than 1.3 fold (less than by 30 %), preferably 1.2 fold (less than by 20 %), more preferably 1.1 fold (less than by 10 %) upon cultur- ing according to the invention, preferably after 5 days or further culturing, in comparison with 10 % (v/v) FCS supplementation.
- an osteoblast direction differentiation occurs in the cells upon culturing according to the invention, preferably after 5 days or further culturing, in comparison with 10 % (v/v) FCS supplementation.
- the expression of at least one preferably two osteoblast specific marker gene(s) is/are in- creased after 5 days incubation in a medium supplemented with SPRF, preferably SPRF having the concentration range given above, preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium as follows:
- - COL1A1 expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold,
- - ALPL expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold, and prefera- bly - RUNX2 expression is at least not reduced or is increased at least 1.05 fold (by 5%), preferably at least 1.1 fold or 1.2 fold,
- the BAX/BCL2 ratio was elevated at least 15, preferably at least 20 or 25 fold both in case of 10 % (v/v) FCS + 1 ng/mL bFGF supplement and in case of the medium as used in the present invention, in particular when 10 % (v/v) SPRF supplementation was used, in comparison with 10 % (v/v) FCS supplementation.
- the invention also relates to a medium or the use of a medium as disclosed herein for increasing proliferation rate of mesenchymal stem cells (MSCs) or for culturing MSCs as disclosed herein.
- the invention also relates to a use of SPRF as a cell medium supplement instead of PRP and FBS wherein said SPRF enhances the proliferation rate of human mesenchymal stem cells in vitro while phenotypi- cal changes were not observed except that the levels of osteoblast markers are increased and differentiation potential of proliferated MSCs was maintained.
- Said medium comprises SPRF as a supplement and as a serum-derived product.
- the medium does not comprise fetal bovine serum (FBS or fetal calf serum, FCS) and does not comprise platelet rich plasma (PRP) and preferably does not comprise FGF (e.g. bFGF) and preferably does not comprise any other growth factor either.
- FBS fetal bovine serum
- PRP platelet rich plasma
- FGF e.g. bFGF
- the medium comprising SPRF does not comprise any further serum product or serum derived product or supplement and preferably does not comprise any other growth factor besides those present in SPRF.
- one or more amino acid source preferably at least glutamate source or a glutamate source only
- one or more salts said salt being preferably selected from calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and monosodium phosphate,
- one or more sugar preferably at least glucose or glucose only and optionally or if desired one or more vitamins preferably selected from folic acid, nicotinamide, riboflavin and B12, wherein said medium comprises 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF.
- the medium may comprise further additives e.g. buffer(s), antibiotic(s), selection agent(s), preservation agent(s) etc.
- the medium is a derivative of Dulbecco' s modified Eagle's medium (DMEM) which differs from DMEM in that it is supplemented with 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.
- DMEM Dulbecco' s modified Eagle's medium
- the SPRF selectively increases mesenchymal stem cell (MSC) proliferation rate which means that proliferation rate of mesenchymal stem cells (MSC) increases at a higher extent than at least one other adult stem cell type, e.g. that of hematopoietic stem cells.
- MSC mesenchymal stem cell
- the invention also relates to the use of serum fraction of platelet rich fibrin (SPRF) obtained from a donor subject to test selective increase of MSC proliferation rate in vitro.
- MSCs are undifferentiated cells with the potential to differentiate in several cell types.
- MSCs doesn't differentiate in vitro into adipocyte direction.
- Said SPRF is a serum fraction of platelet rich fibrin prepared or is obtainable by the following method: SPRF is isolated from whole blood obtained from donors by centrifugation
- SPRF is obtained as an exudate or releasate of the fibrin clot (platelet rich fibrin preferably specifically prepared as disclosed herein).
- SPRF can be collected and stored eg. frozen.
- the SPRF is separated by pressing, squeezing, filtering and/or centrifuging the fibrin clot to isolate the serum fraction (fluid fraction) of platelet rich fibrin.
- the acellular or clear supernatant from the PRF may be isolated, or may be removed before fractionating the PRF to isolate the PRF fluid fraction.
- Such fluid fraction turned out to contain a high concentration of activated platelet releasate and growth factors contained therein.
- the SPRF may be obtained from a blood sample from a single donor subject or from multiple donor subjects and mixed together to obtain a single blood sample.
- SPRF obtained from multiple donors can be mixed together.
- the SPRF is obtained from venous blood collected from a single donor.
- the donor is the patient to whom, once proliferated, the MSCs are reintroduced.
- the SPRF is employed herein without exogenous anticoagulants that are commonly used in the prior art when preparing PRP, thereby an effective activation of platelets and a content of an activated platelet releasate in the isolated serum fraction is obtained according to the invention.
- SPRF is obtained or obtainable by the following method:
- the fibrin clot (platelet rich fibrin) is obtained wherein the red blood cells discarded.
- the SPRF is pressed or squeezed out of the fibrin clot
- the SPRF is collected and stored.
- SPRF is stored frozen or lyophilized, e.g. at -20 °C.
- the invention also relates to the use of SPRF in stem cell therapy preferably in MSC therapy, wherein
- SPRF obtained from a donor subject is used to increase proliferation rate of the patient's in vitro expanded MSCs.
- the donor subject of the SPRF is the same subject as the patient to be treated with MSCs.
- the donor subject of the SPRF is the same subject as the patient from whom the MSCs are obtained.
- SPRF MSCs do not differentiate in vitro.
- osteoblast mark- ers appear on the MSCs on a given period of time e.g. 5 days culturing.
- the invention also relates to the use of SPRF in stem cell therapy wherein the patient's own cells are proliferated in situ (in vivo) or in vitro or ex vivo.
- the patient is a subject in need of bone tissue regeneration.
- the patient is a subject in spongy bone tissue defect, osteonecrosis osteoarthrosis or osteoarthritis.
- the lack of differentiable MSCs in the subchondral/spongy bone can be cured/treated by stem cell therapy or stem cell transplantation.
- the MSC transplantation is autologous transplantation followed by ex vivo multiplication.
- the ex vivo multiplication is carried out in a medium comprising SPRF as the patient' s own blood separation product.
- transplantation is not needed, because the proliferation of resident MSCs can be enhanced.
- the used therapy comprises the proliferation of MSCs resident in a tissue of a patient wherein the SPRF is administered to the tissue of said patient to enhance proliferation of MSCs in said tissue.
- the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.
- the SPRF is administered to the patient to the same site as in vitro expanded own MSCs, essentially simultaneous with or after MSC transplantation.
- the tissue is impaired bone tissue or cartilage tissue.
- SPRF is administered to the patient by matrix assisted transplantation.
- the invention also relates to a method of treatment wherein MSCs are obtained from said patient and the patient's own cells are proliferated in vitro, wherein the MSC transplantation is autologous transplantation followed by ex vivo expansion.
- the invention also relates to a method of treatment of a patient in a stem cell therapy wherein SPRF obtained from a donor subject used to increase proliferation rate of the patient's MSCs expanded ex vivo, wherein the MSCs so proliferated maintain their undifferentiated character with the potential to differentiate in several cell types.
- the MSCs show increased expression of at least one or two, preferably two osteoblast markers, preferably COL1A1 and/or ALPL.
- the donor subject of blood from which the SPRF is obtained is identical with the patient.
- the patient is treated with a condition wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.
- the patient is in need of bone tissue regeneration.
- subchondral and/or spongy bone is treated in a stem cell therapy or stem cell transplantation by MSCs proliferated using said SPRF.
- said therapy comprises the proliferation of MSCs resident in a tissue of a patient wherein the SPRF is administered to the tissue of said patient to enhance proliferation of MSCs in said tissue, and
- the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.
- the tissue is impaired bone tissue or cartilage tissue.
- SPRF is administered to the patient in a matrix.
- subject as used herein shall refer to a warm-blooded mammalian, particularly a human being.
- medical use of the invention or the respective method of treatment applies to a subject in need of administration of a pool of MSCs.
- patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
- treatment is thus meant to include both prophylactic and therapeutic treatment, in particular a treatment including the step of proliferation of MSCs in vitro.
- Platinum rich plasma is a blood fraction prepared by separating the red blood cell fraction from a venous blood sample, removing the red blood cell fraction and, if appropriate, the buffy coat, obtaining thereby a platelet poor plasma fraction (PPP), separating - preferably by centrifugation - a platelet rich fraction from the PPP or pelleting platelets, and recovering the platelets in a platelet rich plasma (PRP) fraction, optionally by resus- pending the pelleted platelets in an appropriate medium, optionally in PPP.
- PPP platelet poor plasma fraction
- Platelet rich fibrin is clotting spontaneously during its preparation by centrifuging a blood sample, preferably accelerated upon contact with negatively charged surfaces and without adding exogenous coagulation activators.
- SPRF serum of platelet rich fibrin
- serum fraction as defined or described in WO2014126970A2.
- SPRF serum of platelet rich fibrin
- SPRF is isolated from whole blood obtained from donors by centrifugation to obtain a fibrin clot and wherein the fibrin clot (platelet rich fibrin) is pressed or squeezed to obtain the SPRF as an exudates or releasate of the fibrin clot.
- SPRF said centrifugation to obtain the fibrin clot is carried out at 1000 to 4000 g, preferably 1000 to 3000 g or 1500 to 2500 g or 1000 to 2500 g or 1500 to 2000 g or more preferably 1500 to 2000 g for 2 to 20 minutes or 3 to 15 minutes or 5 to 15 minutes or 3 to 12 minutes or e.g. 5 or 10 mins, within 4, 3, 2 or preferably within 2 or 1.5 or within 1 minute(s) from blood collction, and SPRF can be collected and stored eg. frozen.
- the coagel is separated by pressing, squeezing, filtering and/or centrifuging the coagel to isolate the serum fraction containing the fluid fraction of platelet rich fibrin.
- the acellular or clear supernatant from the PRF may be isolated, or may be removed before fractionating the PRF to isolate the PRF fluid fraction.
- Such fluid fraction turned out to contain a high concentration of activated platelet releasate and growth factors contained therein.
- the SPRF may be obtained from a blood sample from a single donor or from multiple donors and mixed together to obtain a single blood sample or SPRF.
- the SPRF is obtained from venous blood collected from a single donor.
- the donor is the patient to whom, once proliferated, the MSCs are reintroduced.
- the SPRF is employed herein without exogenous anticoagulants that are commonly used in the prior art when preparing PRP, thereby an effective activation of platelets and a content of an activated platelet releasate in the isolated serum fraction is obtained according to the invention.
- Stem cells are undifferentiated or partially differentiated cells with a strong potential to differentiate into several or multiple differentiated cell types and which are also capable of a limited number of cell division to maintain themselves. Thus, stem cells have a limited capability to proliferate and a high potential to differentiate.
- Adult stem cells (“somatic stem cells” or “tissue stem cells”) are partially differentiated stem cells capable of proliferation, self -renewal, production of a large number of differentiated functional progeny, and are capable of regenerating tissue after injury and having a flexibility in the use of these options
- adult stem cells are e.g.:
- MSC Mesenchymal stem cells
- mesenchymal tissue preferably bone, cartilage or adipose tissue in vitro, and preferably are
- CD105, CD73 and CD90 positive do not carry surface markers of blood progenitor cells or heamatopoietic stem cells, and preferably are CD45, CD34, CD14, CDl lb, CD79a es CD19 negative.
- Cell therapy is the transplantation of human or animal cells to a patient to replace or repair damaged tissue.
- MSC therapy is a cell therapy wherein MSCs are administered to a patient having an impaired tissue and wherein said MSCs are differentiated into cells of said tissue or tissue-specific cells or tissue-resident cells in the patient.
- Osteoarthritis is a degenerative disease characterized by erosion of articular cartilage, which becomes soft, frayed, and thinned with eburnation of subchondral bone and outgrowths of marginal osteophytes; pain and loss of function result; mainly affects weight-bearing joints. Osteoarthritis is also called degenerative joint disease, or osteoarthrosis. Osteoarthrosis may be considered as a chronic noninflammatory bone disease variant and also may be a synonym for osteoarthritis. "Spongy bone” is the tissue that makes up the interior of bones; compact bone is the tissue that forms the surface of bones. In long bones, spongy bone forms the interior of the epiphyses.
- Ostonecrosis is bone death in particular caused by poor blood supply.
- FIG. 1 Schematic comparison of exemplary isolation of platelet rich plasma (PRP) and serum from platelet rich fibrin (SPRF).
- PRP platelet rich plasma
- SPRF platelet rich fibrin
- FIG. 1 Time-course effect of serum supplements on hMSCs.
- Subconfluent hMSCs were cultured in DMEM in the absence of supplement ( ⁇ , col. 1 at day 2 and 5), 10% (v/v) of FCS ( ⁇ , col. 2 at day 2 and 5) or 10% (v/v) FCS + 1 ng/mL bFGF (3 ⁇ 4 col. 3 at day 2 and 5), or 10% (v/v) PRP ( ⁇ , col. 4 at day 2 and 5) OR 10% (v/v) SPRF ( ⁇ , col. 5 at day 2 and 5).
- Results are presented as means of triplicate sapmples in three experiments + SD. p ⁇ 0,0001 ***.
- FIG. 3 Cell morphology of hMSCs using phase contrast microscopy (magnification x 10).
- 3.A 10% (v/v) of FCS (upper row, left);
- 3.B 10% (v/v) FCS + 1 ng/mL bFGF (upper row, right),
- 3.C 10% (v/v) PRP; (lower row, left)
- 3.D 10% (v/v) SPRF (lower row, right).
- FIG. 4 Cell immunophenotypes of hMSCs cultured in differently supplemented media.
- Mesenchymal stem cells were cultured in (4.A) 10% (v/v) of FCS (1) or (4.B) 10% (v/v) FCS + 1 ng/mL bFGF (3 ⁇ 4, or (4.C) 10% (v/v) PRP ( ⁇ ) or (4.D) 10% (v/v) SPRF (SS) and stained with specific antibodies.
- 4.A 10% (v/v) of FCS (1) or (4.B) 10% (v/v) FCS + 1 ng/mL bFGF (3 ⁇ 4, or (4.C) 10% (v/v) PRP ( ⁇ ) or (4.D) 10% (v/v) SPRF (SS) and stained with specific antibodies.
- 4.C 10% (v/v) PRP ( ⁇ ) or (4.D) 10% (v/v) SPRF (SS)
- FACS diagrams representative images of expression of hematopoietic markers CD34, CD l ib, CD 19 and CD45 is shown.
- the fluoro- chromes applied were, respectively, FITC (fluorescein isothiocyanate), Cy5 cyanine dye, APC (Allo- phycocyanin) and PE (Phycoerythrin).
- FIG. 5 Gene expression analysis of differently supplemented hMSCs cultures - On this figure hMSC, adipocyte, osteoblast and apoptotic marker levels are shown in hMSC cultures after 5 days culturing. Mesenchymal stem cells were cultured in 10% (v/v) of FCS (85; background color, control) or (5.B)
- FIG. 7 Histological analysis of hSBPs Culturing hSBPs in 10 % (v/v) SPRF supplemented medium for 5 days preserved bone marrow integrity as hematoxylin and eosin -stained sections (A) and Masson's tri- chrome sections (B) show. However, 10 % (v/v) FCS supplementation for 5 days appeared less effective therein. That means, SPRF revealed higher level of hMSC accumulation (C) compared to FCS (G), and preserved better the local vasculature (D,H).
- FIG. 8 Gene expression analysis of human subchondral bone chips - Relative gene expression level on Y axis is shown compared to the values measured right after the bone chip explantation. .
- B indicates that the hematopoetic cells were not induced, however they could be present in bone chips.
- MSCs are believed to be responsible for replacing cells that are lost in diseases or pathological conditions. Due to these functions the approach of supplementing stem cells to enhance tissue regeneration and treat degenerative diseases were also successfully tested and were shown to be effective. MSCs are also responsible for therapeutic effects in the musculoskeletal system, and were found to be effective in periodontal tissue and bone damage caused by e.g. osteonecrosis and has been successfully applied in cartilage and long bone repair.
- stem cells may alternatively be redistributed using our method, which basically enables selective proliferation of the available stem cells, which means that the proliferative effect can be localized and thus a selective tissue repairing treatment can be realized.
- we focus on local therapeutic effect which means that musculoskeletal and degenerative bone and joint diseases are the main therapeutical targets. This solves the majority of the circulation problems as the circulation of these parts of the body is limited thus the effect of the enhanced proliferation of the stem cells is concentrated on local tissue repair. In our case these tissues are mainly musculoskeletal tissues, more specifically bone and joint tissue.
- the MSCs preserve their stem cell character during the first 5 days of culturing as no differentiation occurs into adipocyte direction with the use of our SPRF culture supplement except the increase in osteoblast factors after 5 days of culture or further culturing. This enables the advantage of not interfering with the MSCs, thus the MSCs will only differentiate as an effect of the surrounding cells at site of the treatment. This gives the opportunity that the stem cells will differentiate in a manner that accelerates the regeneration of the treated tissue.
- SPRF showed surprisingly consequently better results than PRP and similar to or even better than the gold standard cell medium supplement FBS plus growth factors.
- FBS obviously is not appropriate for medicine and is not advisable in cell cultures in transplantation applications.
- MSCs are available from various sources it appears the present invention is not limited to BM derived MSCs and also for example adipogen derived MSC-s could be applied.
- Literature opinions vary in as- sessing the capabilities of MSCs of various sources, an advantage of the present invention may be that due to culturing as disclosed herein multiple sources may become useful and available.
- MSCs are obtained from a subject and said MSCs are cultured by an in vitro method, as disclosed in the Brief Description of the Invention.
- MSC culturing conditions can be applied, for example a DMEM basal medium with sugar source like high glucose, glutamate source like GlutaMaxTM Supplement, pyruvate and antibiotics or selective agents like penicillin/streptomycin and 1% amphotericin.
- DMEM basal medium with sugar source like high glucose, glutamate source like GlutaMaxTM Supplement, pyruvate and antibiotics or selective agents like penicillin/streptomycin and 1% amphotericin.
- FBS regularly used in media like DMEM
- SPRF and only SPRF are to be uses. In a particular embodiment even no further growth factors are to be used.
- OA Osteoarthritis
- Administration of SPRF is conveniently carried out by injection at the site of impaired bone or chondrocyte tissue.
- multiple injection can be applied. For example, injection can be added regularly, e.g. every day or in every 2 days or 1, 2 or 3 times a week.
- SPRF e.g. matrix assisted administration
- hMSC Human mesenchymal stem cell
- hMSCs Human mesenchymal stem cells purchased from LONZA were seeded at 5000 cells/cm 2 in normal T-75 tissue culture flasks and maintained in standard growing medium: Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAXTM Supplement, pyruvate (Gibco, Paisley, Scotland), supplemented with 10 % (v/v) fetal calf serum (FCS, Gibco, Paisley, Scotland), fibroblast growth factor (bFGF) 1 ng/ml (Sigma- Aldrich, St. Louis, USA), 2% Penicillin/Streptomycin (Sigma- Aldrich, St. Louis, USA) and 1% Amphotericin (Sigma- Aldrich, St. Louis, USA). Cell culture medium was refreshed twice a week.
- DMEM Dulbecco's modified Eagle's medium
- FCS Gibco, Paisley, Scotland
- FCS fetal calf serum
- bFGF fibroblast
- Basal medium DMEM, high glucose, GlutaMaxTM Supplement, pyruvate containing 10% FBS, 2% penicillin/streptomycin and 1% amphotericin.
- Serum-free medium DMEM, high glucose, GlutaMaxTM Supplement, pyruvate (Gibco, Paisley, Scotland), containing 2% penicillin/streptomycin and 1% penicillin/streptomycin.
- SPRF-medium DMEM, high glucose, GlutaMaxTM Supplement, pyruvate containing 10% SPRF, 2% penicillin/streptomycin and 1% amphotericin.
- PRP-medium DMEM, high glucose, GlutaMaxTM Supplement, pyruvate containing 10% PRP, 2% penicillin/streptomycin and 1% amphotericin. Media were changed every 48 hours (support for 2 days injection). Cells were incubated in normal cell culture conditions (5% CO 2 , humidified atmosphere, 37 °C). 2000-3000 cells/well were seeded in 5 parallel wells for all sample type and for all time point. Percentage ratios are given in respect of the total volume.
- hSBEs 2 mm in diameter were harvested from patients undergoing total hip replacement surgery at the Orthopedic Clinics of Semmelweis University (Budapest, Hungary). All procedures were performed with permission of hungarian Ethical Committee. The donors had osteoarthritis, otherwise they were diagnosed not to have cancer, or any infectious or autoimmune disease. Only tissue that would have otherwise been discarded was used.
- femoral heads (those would have been otherwise discarded) are sawn off that results in intense cell death at the sawn surface due to friction based heat shock. Therefore, bone explanted from the body was cut in half with a bone chisel and hSBEs were picked from the cut surface with a small chisel.
- Explants were delivered to the laboratory in the same medium that was used for hMSC culture.
- tissue cultures were maintained in basal medium (DMEM containing 10% FBS and 1% penicillin/streptomycin) at 37 °C and 5% CO 2 in a humidified atmosphere. After 2 days incubation samples were used for experiments with special media or harvested for R A.
- basal medium DMEM containing 10% FBS and 1% penicillin/streptomycin
- hMSCs and hSBEs were determined using Cell Proliferation Kit II (XTT; Roche, Mannheim, Germany) according to the manufacturer's instructions. Absorbance were measured after 4 hours incubation in the staining solution using a PowerWave Microplate Spectrophotometer (BioTek, Winooski, VT, USA) at 480 nm with a reference wavelength at 650 nm. In case of hSBEs, bone pieces were removed from the labeling mixture right before absorbance measurement. Results were normalized with the weight of the chips considering that the bone size is proportional to the number of active cells.
- Basal medium DMEM containing 10% FBS and 1% penicillin/streptomycin.
- Serum-free medium DMEM containing 1% penicillin/streptomycin.
- SPRF-medium DMEM containing 10% SPRF and 1% penicillin/streptomycin.
- XTT sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium)
- XTT Labeling Mixture was added to the culture medium and bones or cell monolayers in 0,3 mg/ml final concentration on a 96-well plate and the plate was placed back into the incubator for 4 hours. Bone pieces were removed and absorbance was measured on 450 nm on a plate reader. In case of hSBPs absorbance values were normalized to the dry weight of the bone pieces.
- DEPC diethylpyrocarbonate-treated
- RNA yield was performed by agarose gel electrophoresis and using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The purity of RNA was accepted, when values A260 >2.0 and A260 >2.0 were measured.
- Reverse transcription was performed using the first- strand cDNA synthesis kit as instructed by the manufacturer (ReadyScriptTM, Sigma Aldrich), i.e. reverse transcription to synthesize first strand cDNA was carried out for 30 min at 42 °C, primed with an oligo (dT) primer bearing a T7 promoter.
- Real-time quantitative PCR was performed using ABI for quantifying the expression of activated leukocyte cell adhesion molecule (ALCAM/CD166: Hs00977641_ml). Values were calculated using the comparative threshold cycle (C t ) method and normalized to GAPDH (Hs02758991_gl) expression. Values were expressed as the mean +SD. Experiments were performed at least three times. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey-Kramer Multiple Comparison post-test.
- ANOVA analysis of variance
- Statistically significant differences at p ⁇ 0.05 were determined by one-way analysis of variance (ANOVA).
- Quantitative reverse transcription -polymerase chain reaction analysis was used to evaluate the expression of the hMSC associated gene CD166 (ALCAM, Figure 9). Compared to the second day expression of CD166 (AL- CAM) molecule expression was 2,2-times higher in case of basal medium, and 2,1-times higher in case of SPRF- medium. All expression values are normalized to the expression of GAPDH.
- Our SPRF supplemented medium was as effective as the one, which was supplemented with specific stem cell media (FBS supplemented basal me- dium), however SPRF is from human autologous origin. All expression values are normalized to the expression of GAPDH.
- hMSC cultures were incubated for 2 or 5 days in serum-free DMEM (SF) or in DMEM supplemented either with FCS, FCS + bFGF, PRP or SPRF, 10 % (v/v) each. Viability of the samples was measured with XTT assay on the 1 st , 2 nd and 5 th day of the experiment.
- FCS + bFGF caused an intense (14.18-times) cell number elevation.
- FCS and PRP reached similar viability to FCS + bFGF for the 5 th day, 11.83-fold and 15.53-fold, respectively.
- SPRF SPRF in the medium, where the cell viability 20.1-fold higher was compared to the zero day. This effect is very remarkable considering that the official culture medium and a treatment with a growth factor were less effective (Fig. 2).
- the morphology of the cells was not visibly altered by the various treatments, all preserved the typical hMSC morphology (Fig. 3).
- hMSCs cultured 5 days long in differently supplemented media preserved their hMSC-characteristic.
- hMSCs cultured in 10% (v/v) FCS or 10% (v/v) FCS + 1 ng/mL bFGF, or 10% (v/v) PRP or 10% (v/v) SPRF were positive for CD90-FITC, CD105-PerCP-Cy5.5 and CD73-APC in more than 93.94%.
- CD90-FITC and CD73-APC expression was above 99% in case of the PRP-supplemented samples (Fig. 4.A/1 and /3).
- CD105-PerCP-Cy5.5 level decreased by 13.99% and two cell populations were appeared. This thought to be the result of an unknown differentiation process in connection with PRP (Fig. 4.A/2).
- Panel A shows that mesenchymal stem cell marker levels remained compared to standard culture medium (10 v/v% FCS). On the Y axis the protein expression level ratio is presented, the base of comparison is the level in 10% FCS cultures.
- MSCs may differentiate in either to osteoblasts or adipocytes, consequently both cell type markers have been checked to complete the study.
- panels B and C it is shown that PPARGG and ADIPOQ expression level did not show any change in contrast with osteoblast markers ALPL and COL1A1 which have been significantly increased in case of SPRF supplementation.
- Panel D Apoptotic genes' expression were analyzed as well: BCL2 is antiapoptotic, while BAX is an apoptotic protein. BAX/BCL2 ratio is in equilibrium under normal physiological conditions (taken as 100% in FCS). Increase is demonstrable if cells undergo apoptotic process due to inner or under external effects. FCS+BFGF and SPRF supplementation results in an approximate 80-90%, while PRP cultured cells show a tremendous increase.
- hMSC-specific genes stayed intensely expressed in culture of mesenchymal stem cells supplemented with 10 % (v/v) SPRF
- hMSC-specific genes was confirmed by real time qPCR after 5 days incubation in the media indicated above.
- ALCAM (CD166), ITGB l, CD105 and ANPEP expression showed significant increase in the 10 % (v/v) SPRF supplemented samples when compared with 10 % (v/v) FCS supplemented group 1.68- fold, 2.03-fold, 1.29-fold and 1.37-fold, respectively.
- hMSCs are the common progenitor cells of adipocytes and osteoblasts
- adipocyte-specific and os- teoblast-specific gene expression was investigated in the variously supplemented hMSC cultures by RT-qPCR.
- FABP4, PPARG and ADIPOQ expression that are markers of adipogenic differentiation were not elevated when 10 % (v/v) FCS supplementation was changed for 10 % (v/v) FCS + 1 ng/mL bFGF, 10 % (v/v) PRP or 10 % (v/v) SPRF, i.e. the expression level stayed unvaried compared to standard culturing method (Fig.3B).
- COL1 Al, ALPL and RUNX expression was slightly changed in cultures supplemented with 10 % (v/v) FCS + 1 ng/mL bFGF, 0.78-fold, 1.3-fold and 1.49-fold respectively.
- 10 % (v/v) PRP had almost the same effect 1.43- fold change in COL1A1 expression, 3.11-fold change in ALPL expression and RUNX2 expression decreased 0.47-fold compared to the control group.
- BAX/BCL2 ratio was highly increased when hMSC culture supplemented with 10 % (v/v) PRP BAX/BCL2 ratio was elevated 29.97-fold in case of 10 % (v/v) FCS + 1 ng/mL bFGF supplement, 31.99-fold when 10 % (v/v) SPRF supplementation was used.
- Bone explants were fixed in 4% formalin solution. The samples were dehydrated in an ascending alcohol series at room temperature and infiltrated and embedded in a resin specifically developed for mineralized tissues (Technovit 9100 Kulzer). Infiltrated explants were placed in specific molds filled with polymerization mixture. 4 m-sections were cut using Leica RM2255 sawing microtome and stretched on slides. For hematoxylin-eosin stain staining the sections were immersed into hematoxylin solution and washed with 1% eosin solution. For Mas- son's trichrome staining sections were immersed in hematoxylin solution containing picric acid. After washing, Fuchsin Ponceau staining was performed, and unspecific parts were washed with with 1% phosphomolybdic acide solution.
- Figure 8/B indicates that the hematopoetic cells were not induced, however they could be present in bone chips. This indicates the selectivity of the MSC proliferation method (B/l: CD34 B/2: CD14 B/3: PTPRC). Furthermore, Figure 8/C shows that adipocites were not induced in PSRF medium, thus, similarly to the MSC- cultures, adipocytes differentiation does not occur on explants either (C/l: PPARg C/2: FABP4 C/3: ADPOQ).
- the present invention is applicable both in research and medicine among others to improve proliferation of MSCs with maintaining their proliferation potential preferably for the purpose of bone regeneration or for using them in MSC cell therapy.
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