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Cartilage and bone repair composition.

A technology of cartilage and bone formation, applied in the direction of drug combination, bone/connective tissue cells, microorganisms, etc.

Inactive Publication Date: 2007-08-08
UNIV DE MALAGA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This also avoids the adverse effects of systemic or local infection of bioactive agents, as their control outside the repair site is currently difficult and their pleiotropic effects are an inevitable threat

Method used

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  • Cartilage and bone repair composition.
  • Cartilage and bone repair composition.
  • Cartilage and bone repair composition.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 - Design and acquisition of human fusion recombinant protein TGF-β1 (rhTGF-β1-CBD) with collagen I binding domain

[0034] The cDNA of TGF-β1 was obtained by RT-PCR using a proofreading-capable DNA-polymerase. The template used was total RNA, extracted from human osteosarcoma cells MG63 (ATCC reference: CRL-1427) by the method of Chomczynski and Sacchi (1987). Primers were designed according to the mRNA sequences published in Genbank with the help of software (Oligo v6.6). PCR products were cloned "in dull" in a maintenance plasmid (pBSK II, Stratagene). Once the gene has been cloned, it is tested by double-strand sequencing. Using these plasmids as templates, the region encoding the mature peptide was amplified by PCR with pfu-DNA-polymerase. The primers used contained the collagen binding domain (CBD) and restriction sites suitable for directional cloning in the transfer plasmid of the baculovirus expression system, and contained insect cell-specific sequ...

Embodiment 2

[0041] Example 2.-Obtaining and preparing human mesenchymal stem cells

[0042] Human mesenchymal stem cells were obtained from human bone marrow (BM) following surgical removal of the iliac crest from an informed donor. Bone marrow tissue was cultured in complete medium (-MEM, GIBCO , HD, US). Suspensions of individualized cells were obtained after successive dispersions through 18-, 20- and 22-gauge needles and finally filtered through a 20 micron Teflon sterile filter (Cell Strainer, Falcon, Lincoln Park, NJ) for tissue isolation Residues and cell buildup.

Embodiment 3

[0043] Example 3.- MSC Culture in Collagen Gel

[0044] The cell suspension obtained according to Example 2 was centrifuged at 100 g for 5 minutes. The centrate was resuspended in medium containing very low concentration of patient serum (HS, 0.5%).

[0045] Before the cells were placed in the wells of a 48-well culture plate, 100 mL of a human collagen I solution (Sigma-Aldrich), pH 7.4, at a concentration of 0.35 mg / mL, was placed at the bottom thereof. Mix 150 mL per well of a mixture of collagen I and TGF-β1 at a concentration of 1 mg / mL, and add 2 × 10 6 bone marrow cells on top. The culture plates were placed in a 37°C incubator for 15 minutes to allow the collagen, which was liquid before that time, to form a collagen gel. Then 10 mL of culture medium was placed in each well, also containing the same concentration of TGF-β1. Place the culture thus prepared in 95% air and 5% CO 2in an atmosphere of 37°C and 100% relative humidity. Every 3 days, the medium was chang...

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Abstract

The invention relates to a cartilage and bone repair composition comprising a group of human mesenchymal stem cells that are differentiated to the chondro-osteogenic lineage, by means of the amplification thereof with human serum and a transforming growth factor-Beta 1(TGF-Beta1-DUC) with a molecular domain for binding to collagen I obtained in eukaryotic expression systems, and a biocompatible material which is absorbed by the cells thus treated. The inventive composition can be employed using implants in the area to be repaired or it can be employed directly by injecting the cells in suspension either at the site of the injury or into the systemic circulation for the widespread distribution thereof.

Description

technical field [0001] The present invention can be applied in the biomedical industry involved in the manufacture of drugs involved in bone repair, more specifically in cartilage and bone repair processes of any etiology, spinal fusion or arthrodesis, osteosynthesis, for arthroplasty In surgical prostheses, osteoporosis, etc., and any other situation where bone or cartilage tissue repair or regeneration is required, and where a supplementary supply of differentiated cells is effective in achieving the tissue repair involved. Background technique [0002] Tissue regeneration is a complex process that involves the priming of a variety of cells that are present or have recovered at the site of injury. All events that occur during repair are governed by multiple interactions between growth factors and cytokines, cells and molecules that form the extracellular matrix. [0003] Various inducer signals and growth factors including TGF-β, PDGF, BMP, IGF and FGF co-exist in bone (B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06A61K38/18A61L27/38A61K38/39C12N5/077A61K35/12A61K48/00A61P19/00
CPCC12N2501/15A61L27/3895C12N5/0655A61L27/12A61L27/3817A61L27/3847C12N5/0654A61K35/12A61L27/3821A61K38/1841A61L27/3852C12N2533/54A61P19/00A61P19/08A61K2300/00C12N5/0662A61K38/18A61K38/39
Inventor J·贝希卡拉提亚J·A·安德瑞德斯古美兹M·希弗恩提斯卢达E·古尔拉多帕拉
Owner UNIV DE MALAGA
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