Cartilage-derived morphogenetic proteins

Inactive Publication Date: 2007-03-08
LUYTEN FRANK P +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0008] One aspect of the present invention is a purified cartilage extract that stimulates local cartilage formation when combined with a matrix and implanted into a mammal. This extract can conveniently be produced by a method which includes the steps of: obtaining cartilage tissue; homogenizing the cartilage tissue in the presence of chaotropic agents under conditions that permit separation of proteins from proteoglycans; separating the proteins from the p

Problems solved by technology

One deficiency of the bone induction assay regards its inability to distinguish the physiological roles of different BMP family members.

Method used

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  • Cartilage-derived morphogenetic proteins
  • Cartilage-derived morphogenetic proteins
  • Cartilage-derived morphogenetic proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Characterization of Cartilage Derived Chondrogenic Activity

[0028] Articular (metatarsophalangeal joints), scapular and nasal cartilage (300 grams wet weight per tissue) were prepared from newborn calves. Epiphyseal cartilage was dissected from fetal bovine femurs (7-8 months). The tissues were finely minced and homogenized with a Polytron (top speed, 2×30 seconds) in 20 volumes of 1.2 M GdnHCl, 0.5% CHAPS, 50 mM Tris-HCl pH 7.2, containing protease inhibitors and extracted overnight at 4° C. as described by Luyten et al., in J. Biol. Chem. 264:13377 (1989), which is hereby incorporated by reference. The disclosure of this article is hereby incorporated by reference. Sorgente et al., (Biochem Biophys. Acta. 1972 282:441) disclosed these procedures extract >90% of the lower molecular weight matrix while leaving most of the high molecular weight proteoglycans behind. The extracts were concentrated and exchanged with 6 M urea by diafiltration using an Ultrasette™ (Filtron Technology In...

example 2

PCR Amplification of cDNAs Encoding Cartilage-Derived Morphogenetic Proteins

[0034] Total RNA from bovine articular chondrocytes (metatarsophalangeal joints) was extracted using a modified acid guanidine-phenol-chloroform method described by Chomczynski et al., in Anal. Biochem. 162:156 (1987) and by Luyten et al., in Exp. Cell. Res. 210:224 (1994). Poly(A)+ RNA was isolated using magnetic beads (PolyATract™, Promega, Madison, Wis.). Four degenerate oligonucleotide primers corresponding to highly conversed motifs in the C-terminal region of the BMPs were used; S1: 5′-GGITGG(C / A)AIGA(C / T)TGGAT(A / C / T)(A / G)TIGC(A / C / G / T)CC-3′ (SEQ ID NO: 1) corresponding to amino acids [GW(Q / N)DWI(I / V)AP] (SEQ ID NO: 2); S2: 5′-GGITGG(A / T)(G / C)(I)GA(G / A)TGGAT(T / C / A)ATI(A / T)G(A / C / G / T)CC-3′ (SEQ ID NO: 3) corresponding to amino acids [GWSEWIISP] (SEQ ID NO: 4); AS1: 5′-A(A / G)A / G)GT(C / T)TG(A / C / G / T)AC(A / G)AT(A / G)GC(A / G)TG(A / G)TT-3′ (SEQ ID NO: 5) corresponding to amino acids [NHAIVQTL] (SEQ ID NO: 6); AS2: ...

example 3

Library Screening

[0038] A 120 bp PCR fragment encoding part of the C-terminal domain of novel B like genes (dashed line, FIG. 1) was used to screen two cDNA libraries. One library, from adolescent human articular cartilage poly(A)+ RNA (kindly provided by Dr. Bjorn Olsen, Harvard, Boston, Mass.), was primed with oligo dT and constructed in the λgtl 1 vector. The other was a bovine oligo dT and random primed articular cartilage cDNA library constructed in the UNIZAP®XR vector (Stratagene, La Jolla, Calif.). Approximately 1×106 plaques from each library were screened by standard procedures. Hybridizations were performed for 20 hours at 42° C. in 6×SSC, 1× Denhardt's solution, 0.01% tRNA, 0.05% sodium pyrophosphate and the membranes (DuPont 137 mm nylon membranes, New England Nuclear, MA) were washed to final stringency of 6×.SSC, 0.1% SDS at 55° C. for 20 minutes.

[0039] Thus, cloned inserts having novel BMP-like sequences were isolated, radiolabeled and used to screen both human and...

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Abstract

Nucleotide and amino acid sequences of cartilage-derived morphogenetic proteins (CDMP-1 and CDMP-2) from human and bovine cartilage extracts. These proteins exhibit chondrogenic activity and can be used to repair cartilage defects in a mammal.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 379,830, filed Mar. 3, 2003, which is a continuation of U.S. application Ser. No. 09 / 730,772, filed Nov. 30, 2000, now abandoned, which is a continuation of U.S. application Ser. No. 08 / 836,081, filed Jul. 28, 1997, now abandoned, which represents the U.S. national phase of International Application No. PCT / US94 / 12814, filed Nov. 7, 1994, designating the United States of America and published in English on May 17, 1996 as WO 96 / 14335.FIELD OF THE INVENTION [0002] The present invention relates generally to the field of cartilage and bone development. More specifically, the invention relates to cartilage-derived morphogenetic proteins that stimulate development and repair of cartilage in vivo. BACKGROUND OF THE INVENTION [0003] Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily that can induce endochondral bone formation in adult animals. This superfam...

Claims

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Application Information

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IPC IPC(8): A61K35/34A61K38/17C12Q1/68C07H21/04C12P21/06A61K35/32A61K38/18C07K14/51C07K14/78
CPCA61K35/32A61K38/1875Y10S930/12C07K14/78C07K14/51
Inventor LUYTEN, FRANK P.MOOS, MALCOLM JR.CHANG, STEVEN CHAO-HUAN
Owner LUYTEN FRANK P
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