WO2005112542A2 - Composición para la reparación ósea y cartilaginosa - Google Patents
Composición para la reparación ósea y cartilaginosa Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/3895—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 containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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Definitions
- the present invention has its application within the biomedical industry dedicated to the manufacture of agents involved in skeletal repair and, more specifically, in the processes of cartilage and bone repair of any etiology, spinal fusions or arthrodesis, osteosynthesis, prostheses' for arthroplasties, osteoporosis, etc., and any others that require repair or regeneration of bone or cartilaginous tissues and in which a supplementary contribution of cells in differentiation supposes an effective aid for the achievement of the tissue repair in question.
- Tissue regeneration is a complex process that involves the culmination of the work of a wide variety of cells that were either present or have been recruited at the site of the lesion. All events that occur in the repair process are guided by a series of interactions between growth factors and cytokines, cells and the molecules that constitute the extracellular matrix.
- inductive signals and growth factors coexist in bone, including TGF- ⁇ , PDGF, BMPs, IGFs and FGFs (Becerra et al. Med Clin 2001; 116: 23-34.). Of all of them, only BMPs are osteoinductors, however all are involved in the repair of a particular fracture (Reddi Nature Medicine 1998, 3: 837-839; Groeneveld and Burger Eur J Endocrinol 2000; 142: 9-21) Also in the bone there are osteogenic precursor cells that respond to the stimulation of the fracture, activating and producing BMPs that induce the migration of mesenchymal stem cells (CMMs), which proliferate and differentiate into bone-forming cells (Guerado et al.
- CCMs mesenchymal stem cells
- TGF- ⁇ s constitute a group of growth factors, encoded by a gene family that is expressed in numerous tissues and species (Kingsley Genes Dev; 1994; 8: 133-146; Bostrom and Asnis, Clin Orthop Relat R, 1998; 355S : 124-131). Bone was one of the first tissues in which local production of a TGF- ⁇ was observed with the ability to regulate cell function (Centrella et al. J Bone Joint Surg 1991; 73-A: 1418-1428). Although the main source of TGF- ⁇ is platelets, bone is the most important deposit, 100 times greater than that of other soft tissues, where there is also a shared presence of BMPs (Roberts et al. Biochemistry 1983; 22: 5692- 5698).
- the transplantation of trained cells should be done on a microenvironment that promotes osteogenesis, when the consolidation of two or more bone fragments is what is required.
- the fracture or non-consolidation focus prseudoarthrosis
- the simple non-dynamic rigid stabilization of the focus by intra or extramedullary implants should promote consolidation.
- the transplantation of trained cells into osteogenic lineage on a carrier ("carrier") of hydroxyapatite or collagen finds a hypervascular and stable bed after osteosynthesis, ideal microenvironment for the development of the osteogenic callus.
- the present invention has an obvious application for the preparation of agents and compositions that serve for skeletal repair, and more specifically in the processes of repairing cartilage, bone, osteoporosis, prostheses for arthroplasties, etc., which can be injected in situ in small fracture that does not join or in cartilaginous lesions, in the systemic circulation of osteoporotic individuals or implanted adsorbed in the appropriate biomaterial (collagen, hydroxyapatite, etc.) to promote the formation of cartilage or bone in lesions of a certain entity. They can also be adsorbed on the hydroxyapatite (periapatite) that covers the prostheses for hip, knee, etc. arthroplasties to facilitate the biological integration of these prostheses in the host, extending their life. These preparations can also be used in spinal fusion to promote spinal fusions and improve the efficiency of such operations performed by the usual procedures with autograft or bench bone.
- the present invention provides a method for increasing bone and / or cartilage formation in a human in need thereof by administering to it human mesenchymal stem cells, differentiated into the chondroosteogenic lineage by their amplification with human serum and a transforming growth factor beta 1 with a molecular domain of binding to collagen I (TGF- ⁇ -DUC) obtained in eukaryotic expression systems.
- human mesenchymal stem cells differentiated into the chondroosteogenic lineage by their amplification with human serum and a transforming growth factor beta 1 with a molecular domain of binding to collagen I (TGF- ⁇ -DUC) obtained in eukaryotic expression systems.
- the administration of said trained cells is carried out by injection in situ, in the systemic circulation or by implants, adsorbed in the appropriate biomaterial.
- it is done through the use of implants, adsorbed cells in the appropriate material, the use of reabsorbable hydroxyapatite, with 50% porosity, diffusion chambers or collagen gels being even more preferred.
- human CMMs have been aided in their induction into the chondroostegenic lineage using a glucocorticoid.
- dexamethasone being preferred.
- this induction is carried out in the presence of dexamethasoxane and ⁇ -glycerophosphate.
- the transforming growth factor beta 1 fused with a molecular domain of collagen binding I has been expressed in eukaryotic expression systems. Expression is preferably carried out using insect cells, and even more preferred is the use of insect cells transfected with baculovirus.
- a method of in vitro training of chondroosterogenic cells from human CMMs which comprises the following steps:
- TGF- ⁇ -DUC transforming growth factor beta 1 with a molecular domain of binding to collagen I
- Human CMMs can be obtained from different tissues and locations of the patient himself, as well as from the bone marrow, umbilical cord blood, peripheral blood or the internal mass of frozen human blastocysts.
- the transforming growth factor beta 1 fused with a molecular domain of binding to collagen I has been expressed in eukaryotic expression systems. More preferably, insect cells are used for expression. The use of insect cells transfected with baculovirus is even more preferred.
- the primary culture is performed on a human type I collagen gel.
- the selection of the bone marrow CMMs is carried out in a culture supplemented with an amount between 0.1 and 1% of human serum, preferably 0.5%.
- the amplification takes place over a period between 1 to 25 days, during which the culture medium is supplemented with an amount between 5% and 25% of human serum, preferably 20%.
- a third aspect of the present invention provides cells with chondroosterogenic capacity obtainable according to the above training procedure, which comprises the selection, amplification and chondroosterogenic induction of CMMs.
- chondroosterogenic capacity cells obtainable in accordance with the above training procedure is provided in the preparation of a composition for bone and / or cartilaginous repair in humans.
- a composition capable of inducing osteogenesis comprising a material is provided biocompatible and osteoconductive, and a set of cells with chondroostegenic capacity obtainable according to the previous training procedure.
- FIG. 1 MO cells cultured in vitro for 10 days in collagen gel, in the presence of rhTGF- ⁇ -DUC. Few isolated cells are observed. MO cells cultured 35 days in collagen gel (b and c), in the presence of rhTGF- ⁇ 1-DUC and induced with dexamethasone and ⁇ -glycerophosphate, respectively. A higher density of cells and some colonies thereof are observed. x60
- Figure 2. In a) radiographs of four diffusion chambers are shown after the implantation period in the subcutaneous tissue of the rat. Different densities indicate different quality and quantity of neoformed tissue. b) Shows a histological section of a chamber after the implantation period, in which cultured cells were placed under control conditions, without rhTGF- ⁇ -DUC; Its interior is full of more or less fibrous material, without the accumulation of chondro-osteoid tissue (asterisk, chamber filter). Syrium-hematoxylin red. xlO.
- x40 e Section parallel to the previous one, stained with Goldner's trichrome showing an intense blue color (empty circle) the densest tissue (osteoid) and a pale color which resembles cartilage (full circle). Surrounding the accumulation appear bluish fibers that seem to constitute a layer of perichondrium (arrows).
- x40 f When another parallel section is colored with Alcian Blue, the cartilaginous tissue appears blue (full circle), while the osteoid appears colorless (empty circle), as well as the perichondrium.
- Figure 3 Sections of tissue formed in the place where hydroxyapatite implants were performed with processed cells as indicated in the text. Since the material was descaled before being cut and colored, the empty spaces (asterisks) correspond to the walls of the hydroxyapatite trabeculae. The gaps between these walls are now filled with a tissue that can be called medullary (full circles), while thin sheets of neoformed tissue (arrows) appear attached to these trabeculae.
- Example 1 Design and obtaining of the recombinant human fusion protein TGF- ⁇ 1 with a collagen I binding domain (rhTGF- ⁇ 1-DUC).
- the cDNA for the TGF- ⁇ 1 gene was obtained by RT-PCR, using a DNA polymerase with correction capability.
- Total RNA extracted by the method of Chomczynski and Sacchi (1987), was used as a template from MG63 human osteosarcoma cells (ref. In ATCC: CRL-1427).
- Primers are designed with the help of software (Oligo v6.6), based on the sequences of mRNAs published in the genbank.
- the PCR products were cloned "in blunt", in a maintenance plasmid (pBSK II, Stratagene). Once the genes were cloned, they were checked by sequencing of both strands.
- plasmids were amplified by PCR with pfu-AND-polymerase, the region that codes for the mature peptide.
- the primers used incorporate the Collagen Binding Domain (DUC) and restriction sites suitable for directional cloning in a transfer plasmid of a baculovirus expression system, which contains a sequence for a cell-specific secretion signal peptide of insect (pAcGP67B, Pharmingen). To obtain the factor without DUC, the primers had only restriction sites.
- DUC Collagen Binding Domain
- the primer for the sense chain had the following structure: 5 '- (BamHI) -sequence of the DUC-GGAGGA- (NheI) -6 first nucleotide mature peptide TGF- ⁇ 1.
- the primer for the chain antisense consisted of several nucleotides at the 5 'end of the gene and that comprised the stop codon. It also had the Not I site. In all cases, the construction was checked by sequencing the PCR product or the transfer plasmid containing it.
- insect cells were co-transfected with a mixture composed of the transfer plasmid and a commercial baculovirus DNA (BacVector3000, Novagen), which has been removed in addition to the polyhedrin gene, other genes that code for cysteine proteases.
- BocVector3000 commercial baculovirus DNA
- liposomes lipofectin, Novagen
- the DNA was transferred to the cells, they were cultured in TNM-FH medium for several days, until affectation was observed in the cells. During this time, homologous recombination occurs inside the cells, transferring viral DNA, "downstream" of the polyhedrin promoter, a region of the plasmid comprising the sequence of the signal peptide plus that of the factor with or without DUC. This process generates infective viruses that kill the cell, are released into the environment and infect other cells.
- a recombinant virus was selected from the mixture of recombinant viruses in the culture supernatant. This was done by plaque assay, which consists of sowing a culture of semi-confluent cells with dilutions of the transfection supernatant. After the infection period (lh), the plate was covered with agarose dissolved in culture medium and allowed to grow for 4 days. This allowed the spread of the infection to occur in the environment of some cells, which generated a lysis plate. These lysis plates were removed individually and deposited in a small volume of medium to extract the viruses.
- this procedure was repeated once again from an anterior lysis plate and This way we will make sure we have a recombinant virus cloned.
- this virus was amplified, it was analyzed by PCR with the specific primers for the factor, to verify that it contained the gene of interest. Next, it was necessary to generate a large volume stock that had to be titled, to know the number of pfu / ml (plate forming units / ml).
- MOI virus / cell number
- the well cells were broken by freezing-thawing and an extract was made with PBS. Both samples were analyzed by SDS-electrophoresis (denaturing conditions) and without reducing agents. The polyacrylamide gel was stained with Coomasie Blue (if it was not sensitive enough, silver staining can be performed). The different samples were also analyzed by immunoblot using antibodies specific for TGF- ⁇ 1. We used a secondary antibody labeled with peroxidase and detected its presence by chemiluminescence (CSPD, Amersham). With this information we evaluate the proportion of monomer / dimer that was produced and the performance of the system.
- SDS-electrophoresis denaturing conditions
- Coomasie Blue if it was not sensitive enough, silver staining can be performed.
- the different samples were also analyzed by immunoblot using antibodies specific for TGF- ⁇ 1. We used a secondary antibody labeled with peroxidase and detected its presence by chemiluminescence (CSPD, Amersham). With this information we evaluate the proportion of
- a liquid culture of approximately 250ml was started, with about 500,000 cels / ml. When reached a growth of about 2x106 cels / ml, was inoculated with the virus, using the highest MOI and whose yield was higher. At 24 hours the medium was removed with serum and the cells were continued to be cultured with serum-free medium, the number of days determined by previous tests. After this time, the proteins were purified according to the protocol described by Aono et al. (nineteen ninety five). The method uses cation exchange chromatography, using a SP-Sepharose column (Amesham-Pharmacia).
- the culture medium was equilibrated to pH 8 and 4M urea was added.
- the column was equilibrated with a 20 mM Tris-HCl buffer pH8, 4M urea. After the medium passed through the column, it was washed with the buffer and the proteins were eluted with a linear NaCl gradient between 0 and 0.7M.
- the absorbance at 280 nm was measured at the different fractions and those that had proteins were analyzed by electrophoresis, following the technique described above.
- the positive fractions were collected and concentrated by ultrafiltration and used in biological activity assays. The protein concentration of this final fraction was measured by Bradford.
- Example 2 Obtaining and preparing human mesenchymal stem cells.
- Human mesenchymal stem cells were obtained from human bone marrow (MO) after surgical removal of the iliac crest from informed donors.
- the spinal tissue was washed in complete medium (D-MEM GD3CO, HD, USA). with antibiotics (100 mg / ml penicillin G (sigma), 50 mg / ml gentamicin (sigma) and 0.3 mg / ml fungizone (Flow-ICN)).
- the individualized cell suspension was obtained after sequential disintegration through 18, 20 and 22 gauge needles, and finally filtered through sterile 20 micrometer Teflon filters (Cell Strainer, Falcon, Lincoln Park, NJ) to separate debris from tissue and cell clusters.
- the cell suspension obtained according to example 2 was centrifuged at 100 g for 5 min. The centrifuge was resuspended in culture medium with very low serum concentration of the patient (SH, 0.5%).
- plates 48 was deposited in the bottom of the same 100 ml. of a solution of human collagen I (Sigma-Aldrich), at a concentration of 0.35 mg / ml. at pH 7.4.
- the culture plates were introduced into the culture oven at 37 ° C, for 15 min. to allow the formation of the collagen gel, which until now liquid was maintained.
- 150 ml were deposited per well of culture medium also containing the same concentration of TGF- ⁇ 1.
- the cultures thus arranged were cultured in an atmosphere of 95% air and 5% CO 2 at 37 ° C and 100 % relative humidity.
- the culture medium was changed every 3 days, adding in each case the same concentration of TGF- ⁇ 1.
- TGF- ⁇ 1 The optimal concentration of TGF- ⁇ 1 was investigated in previous trials of chemotactic response.
- 150 ml of collagen (0.35 mg / ml) was mixed with the concentration of the previously selected factor as the one with the highest chemotactic capacity.
- Confluent rat bone marrow cultures were dissociated and the cells were placed in the upper compartment of the Boyden chambers (Neuroprobe, Rockville, MD) at a concentration of 2x10 5 cells / ml of D-MEM.
- the lower chamber compartment contained the medium with the different concentrations of the factor to be tested.
- the filters were removed and fixed in methanol; they washed, stained and mounted on doors. Chemotaxis was quantified by counting the number of cells that migrated in 20 microscope fields (x400) in each filter.
- Example 4.- Experimental procedure for cell training selection, amplification and induction).
- the MO cells were maintained under the experimental conditions described above for 10 days, changing the culture medium, and therefore TGF- ⁇ 1, every 3 days. After 10 days, the culture medium with 10% of the patient's own serum (SH 10%) was implemented. Thus ending the so-called selection period and the amplification period begins, which was maintained for the time necessary to obtain a quantity of cells that makes transplantation possible, but in any case, this period should not exceed 25 days. After this period, the medium was implemented with dexamethasone (10 " M) and ⁇ -ghcerophosphate (2 mM) that act as osteoinductive chemical agents. This period of only 2 days is called induction.
- Tables I, II, III represent the values obtained. They show how TGF- ⁇ 1-DUC increases the number of cells compared to the control. The same happens with the synthesis of alkaline phosphatase.
- the calcium content is also increasing with the days of cultivation, while in the control the values are imperceptible.
- the data correspond to the mean values and their ⁇ SD for every four samples.
- ND not detected, p ⁇ 0.01 compared to the control cultures.
- the data correspond to the mean values and their ⁇ SD for every four samples.
- Example 5 Transfer of the trained cells.
- the collagen gels were digested with collagenase and the cells were broken down with 0.05% trypsin and 0.02% EDTA, at 37 ° C.
- the cells were centrifuged, washed and resuspended in serum free medium. After the corresponding count, part of them were introduced into Millipore diffusion chambers that were implanted in the rat's subcutaneous tissue, and another part was absorbed into hydroxyapatite fragments that are implanted in a specific bone lesion ( Figure 3).
- Example 6 Subcutaneous implantation. Ectopic bone formation.
- the diffusion chambers with lxl O 6 human cells trained by the described procedure and suspended in serum-free medium were implanted subcutaneously in the back of 344 female Fischer rats 7 months of age, prior anesthesia with subcutaneous injection of sodium pentobarbital 3.5 mg / 100 g body weight. A 2 cm incision was made. in the midline of the back to introduce the cameras.
- Each rat housed several chambers with cells treated with TGF- ⁇ -DUC and other controls, with cells that passed the culture period without treatment or with commercial TGF- ⁇ 1.
- the wounds were sutured and disinfected with Betadine. Four weeks later (implantation period), the rats were sacrificed by anesthetic overdose and the cameras removed for histological and radiographic study.
- the latter was practiced with low intensity X-rays.
- the cameras were fixed in Bounin or 10% Buffered Formalin. After fixing, the plastic ring of the chambers was dissolved by immersion in acetone. After dehydration, they were included in paraffin and cut sagittally at various levels, 5 micrometers thick. Sections were stained by different histochemical or immunocytochemical techniques with antibodies against different types of collagen, with in order to analyze the tissue formed inside the chambers during the implantation period.
- Figure 2 shows radiographic and histological images of different chambers that demonstrate the cartilaginous and bone nature of the tissue formed in them.
- the cartilaginous tissue seems to transform into osteoid-bone, so it seems to indicate that inside the chambers there is a genuine process of endochondral ossification.
- Example 7 Example of Implantation in a tibial fracture. Fragments of Proosteon 500 hydroxyapatite (hiterpore) of about 0.5 cm in diameter were embedded with the cell suspension, dripping on them several microliters, and changing their position to ensure that most of their cavities were filled with cells. The cells thus arranged were transferred to the operating room, taking care of the asepsis and the temperature slightly above 0 ° C. Anesthetized the patient, the fracture surfaces were prepared and hydroxyapatite fragments were introduced separately. This fracture was really a non-union or atrophic pseudoarthrosis, characterized by an avascular focus with fibrous tissue, without proliferation of vascularized chondro-osteogenic tissue.
- Figure 3 shows images of histological sections performed on biopsy after six weeks of implantation. They show the neoformed tissue in contact with the walls of the hydroxyapatite with bone tissue qualities, as well as the formation of spinal tissue in the gaps that remain between the ore trabeculae. The evidence presented shows that the tissue formed has characteristics of bone in the process of maturation. Likewise, the images show that osteoinduction extends near the implants but not much further, which can be explained because the transplanted cells are responsible for such osteoinduction, extending there where the cells spontaneously spread in the implant area. The areas farthest from the implant have no symptoms of osteoinduction.
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
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MXPA06013492A MXPA06013492A (es) | 2004-05-21 | 2005-05-20 | Composicion para la reparacion osea y cartilaginosa. |
AU2005244626A AU2005244626A1 (en) | 2004-05-21 | 2005-05-20 | Cartilage and bone repair composition |
JP2007517309A JP2007538055A (ja) | 2004-05-21 | 2005-05-20 | 軟骨および骨修復用組成物 |
US11/597,236 US20080031915A1 (en) | 2004-05-21 | 2005-05-20 | Cartilage and Bone Repair Composition |
BRPI0510829-2A BRPI0510829A (pt) | 2004-05-21 | 2005-05-20 | uso de células tronco mesenquimáticas humanas, procedimento de capacitação in vitro de células condro-osteogênicas a partir de células tronco mesenquimáticas humanas, células com capacidade condro-osteogênica, uso de células com capacidade condro-osteogênica e composição capaz de induzir osteogênesis |
EP05750573A EP1760144A2 (en) | 2004-05-21 | 2005-05-20 | Cartilage and bone repair composition |
NZ552180A NZ552180A (en) | 2004-05-21 | 2005-05-20 | Cartilage and bone repair composition comprising human mesenchymal stem cells |
CA002567314A CA2567314A1 (en) | 2004-05-21 | 2005-05-20 | Cartilage and bone repair composition |
IL179442A IL179442A0 (en) | 2004-05-21 | 2006-11-21 | Cartilage and bone repair composition |
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ESP200401227 | 2004-05-21 | ||
ES200401227A ES2245879B1 (es) | 2004-05-21 | 2004-05-21 | Composicion para la reparacion osea o cartilaginosa. |
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WO2005112542A2 true WO2005112542A2 (es) | 2005-12-01 |
WO2005112542A3 WO2005112542A3 (es) | 2006-03-23 |
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PCT/ES2005/000287 WO2005112542A2 (es) | 2004-05-21 | 2005-05-20 | Composición para la reparación ósea y cartilaginosa |
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US (1) | US20080031915A1 (es) |
EP (1) | EP1760144A2 (es) |
JP (1) | JP2007538055A (es) |
KR (1) | KR20070047740A (es) |
CN (1) | CN101014701A (es) |
AU (1) | AU2005244626A1 (es) |
BR (1) | BRPI0510829A (es) |
CA (1) | CA2567314A1 (es) |
ES (1) | ES2245879B1 (es) |
IL (1) | IL179442A0 (es) |
MX (1) | MXPA06013492A (es) |
NZ (1) | NZ552180A (es) |
RU (1) | RU2375447C2 (es) |
WO (1) | WO2005112542A2 (es) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007085210A3 (en) * | 2006-01-25 | 2008-01-03 | Univ Karlova | Method of cultivation of human mesenchymal stem cells, particularly for the treatment of non-healing fractures, and bioreactor for carrying out this cultivation method |
US9588121B2 (en) | 2009-11-06 | 2017-03-07 | Go Therapeutics, Inc. | Method for early detection of cancer |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050064042A1 (en) * | 2003-04-29 | 2005-03-24 | Musculoskeletal Transplant Foundation | Cartilage implant plug with fibrin glue and method for implantation |
US7067123B2 (en) | 2003-04-29 | 2006-06-27 | Musculoskeletal Transplant Foundation | Glue for cartilage repair |
US20050222687A1 (en) * | 2004-04-02 | 2005-10-06 | Gordana Vunjak-Novakovic | Cartilage implant assembly and method for implantation |
US7901457B2 (en) * | 2003-05-16 | 2011-03-08 | Musculoskeletal Transplant Foundation | Cartilage allograft plug |
US20080220044A1 (en) * | 2007-03-06 | 2008-09-11 | Semler Eric J | Cancellous construct with support ring for repair of osteochondral defects |
US20090319045A1 (en) | 2004-10-12 | 2009-12-24 | Truncale Katherine G | Cancellous constructs, cartilage particles and combinations of cancellous constructs and cartilage particles |
US7837740B2 (en) * | 2007-01-24 | 2010-11-23 | Musculoskeletal Transplant Foundation | Two piece cancellous construct for cartilage repair |
US7815926B2 (en) * | 2005-07-11 | 2010-10-19 | Musculoskeletal Transplant Foundation | Implant for articular cartilage repair |
EP1926459B1 (en) | 2005-09-19 | 2015-01-07 | Histogenics Corporation | Cell-support matrix having narrowly defined uniformly vertically and non-randomly organized porosity and pore density and a method for preparation thereof |
US8435551B2 (en) * | 2007-03-06 | 2013-05-07 | Musculoskeletal Transplant Foundation | Cancellous construct with support ring for repair of osteochondral defects |
EP2137300B1 (en) | 2007-04-26 | 2011-10-26 | Ramot at Tel-Aviv University Ltd. | Pluripotent autologous stem cells from oral or gastrointestinal mucosa |
US8062364B1 (en) | 2007-04-27 | 2011-11-22 | Knee Creations, Llc | Osteoarthritis treatment and device |
EP2344631B1 (en) | 2008-10-31 | 2019-10-16 | Synthes GmbH | Method and device for activating stem cells |
CN101705208B (zh) * | 2009-12-15 | 2012-05-02 | 北京科润维德生物技术有限责任公司 | 将人羊膜间充质细胞诱导分化成骨或软骨细胞的方法 |
CN102971414B (zh) * | 2009-12-22 | 2016-01-27 | 新加坡科技研究局 | 骨折的治疗 |
US8574614B2 (en) * | 2010-02-04 | 2013-11-05 | Hwa-Chang Liu | Surgical grafts for repairing chondral defects |
RU2456927C2 (ru) * | 2010-10-13 | 2012-07-27 | Федеральное государственное учреждение "Российский научный центр "Восстановительная травматология и ортопедия" имени академика Г.А. Илизарова" Министерства здравоохранения и социального развития Российской Федерации ФГУ "РНЦ "ВТО" им. акад. Г.А. Илизарова | Способ биологической оценки остеоиндуцирующего эффекта белков плазмы крови |
US10077420B2 (en) | 2014-12-02 | 2018-09-18 | Histogenics Corporation | Cell and tissue culture container |
CN107224618A (zh) * | 2016-03-25 | 2017-10-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | 促进骨再生的活性支架材料、其制备方法及应用 |
AU2017204355B2 (en) | 2016-07-08 | 2021-09-09 | Mako Surgical Corp. | Scaffold for alloprosthetic composite implant |
CN108578777B (zh) * | 2018-05-06 | 2021-05-07 | 西北工业大学 | 一种生长因子浓度梯度可控的人工硬骨支架制备方法 |
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- 2005-05-20 CN CNA2005800230591A patent/CN101014701A/zh active Pending
- 2005-05-20 MX MXPA06013492A patent/MXPA06013492A/es not_active Application Discontinuation
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Non-Patent Citations (5)
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007085210A3 (en) * | 2006-01-25 | 2008-01-03 | Univ Karlova | Method of cultivation of human mesenchymal stem cells, particularly for the treatment of non-healing fractures, and bioreactor for carrying out this cultivation method |
CZ301148B6 (cs) * | 2006-01-25 | 2009-11-18 | Univerzita Karlova V Praze | Zpusob pestování lidských mezenchymových kmenových bunek, zejména pro lécbu nehojících se fraktur, a bioreaktor k provádení tohoto zpusobu |
US9588121B2 (en) | 2009-11-06 | 2017-03-07 | Go Therapeutics, Inc. | Method for early detection of cancer |
Also Published As
Publication number | Publication date |
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ES2245879B1 (es) | 2006-12-16 |
US20080031915A1 (en) | 2008-02-07 |
BRPI0510829A (pt) | 2007-11-27 |
RU2375447C2 (ru) | 2009-12-10 |
CN101014701A (zh) | 2007-08-08 |
EP1760144A2 (en) | 2007-03-07 |
AU2005244626A1 (en) | 2005-12-01 |
WO2005112542A3 (es) | 2006-03-23 |
ES2245879A1 (es) | 2006-01-16 |
CA2567314A1 (en) | 2005-12-01 |
RU2006145431A (ru) | 2008-06-27 |
KR20070047740A (ko) | 2007-05-07 |
JP2007538055A (ja) | 2007-12-27 |
NZ552180A (en) | 2009-11-27 |
IL179442A0 (en) | 2007-05-15 |
MXPA06013492A (es) | 2007-01-23 |
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