High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects

被引:57
作者
Goomer, RS
Deftos, LJ
Terkeltaub, R
Maris, T
Lee, MC
Harwood, FL
Amiel, D
机构
[1] Univ Calif San Diego, Sch Med, Dept Orthoped, La Jolla, CA 92093 USA
[2] Univ Calif San Diego, Sch Med, Dept Med, La Jolla, CA 92093 USA
[3] Vet Adm Med Ctr, La Jolla, CA USA
[4] Seoul Natl Univ, Coll Med, Dept Orthoped Surg, Seoul, South Korea
关键词
gene therapy; tissue engineering; articular cartilage repair; transfection; primary perichondrial cells; primary chondrocytes; PTHrP; TGF-beta; 1;
D O I
10.1053/joca.2000.0382
中图分类号
R826.8 [整形外科学]; R782.2 [口腔颌面部整形外科学]; R726.2 [小儿整形外科学]; R62 [整形外科学(修复外科学)];
学科分类号
摘要
Background: Primary perichondrial cells and chondrocytes have been used to repair articular cartilage defects in tissue engineering studies involving various animal models. Transfection of these cells with a gene that induces chondrocytic phenotype may form an ideal method to affect tissue engineering of articular cartilage. Design: A protocol for high-efficiency transfection of primary perichondrial and cartilage cells was optimized. Plasmids carrying the marker beta -galactosidase (beta -gal), PTHrP and TGF-beta1 genes driven by a strong mammalian promoter were transfected into primary perichondrial cells and chondrocytes. A three-step method was used to achieve high efficiency of transfection: (1) permeabilization of primary cells using a mild detergent, (2) association of plasmid DNAs with a polycationic (poly-l-lysine) core covalently linked to a receptor ligand (transferrin), (3) introduction of cationic liposomes to form the quaternary complex. For in-vivo assessment, polylactic acid (PLA) scaffolds seeded with beta -gal transfected perichondrial cells were implanted into experimentally created osteochondral defects in rabbit knees for 1 week. Results: The efficiency of transfection was determined to be over 70% in vitro. The transformed cells continued to express beta -gal, in vivo for the entire test period of 7 days. Furthermore, primary perichondrial cells transfected with TGF-beta1 and PTHrP over-expressed their cognate gene products. Conclusion: The ability to transfect autologous primary perichondrial cells and chondrocytes with high efficiency using a non-viral system may form a first step towards tissue engineering with these transformed cells to repair articular cartilage defects. (C) 2001 OsteoAtthritis Research Society International.
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页码:248 / 256
页数:9
相关论文
共 61 条
[11]   NO LUNG TOXICITY AFTER REPEATED AEROSOL OR INTRAVENOUS DELIVERY OF PLASMID-CATIONIC LIPOSOME COMPLEXES [J].
CANONICO, AE ;
PLITMAN, JD ;
CONARY, JT ;
MEYRICK, BO ;
BRIGHAM, KL .
JOURNAL OF APPLIED PHYSIOLOGY, 1994, 77 (01) :415-419
[12]   Receptor ligand-facilitated gene transfer: Enhancement of liposome-mediated gene transfer and expression by transferrin [J].
Cheng, PW .
HUMAN GENE THERAPY, 1996, 7 (03) :275-282
[13]   ARTICULAR-CARTILAGE REPAIR USING ALLOGENEIC PERICHONDROCYTE-SEEDED BIODEGRADABLE POROUS POLYLACTIC ACID (PLA) - A TISSUE-ENGINEERING STUDY [J].
CHU, CR ;
COUTTS, RD ;
YOSHIOKA, M ;
HARWOOD, FL ;
MONOSOV, AZ ;
AMIEL, D .
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, 1995, 29 (09) :1147-1154
[14]  
Chu CR, 1997, CLIN ORTHOP RELAT R, P220
[15]   CYSTIC-FIBROSIS GENE-THERAPY [J].
COLLEDGE, WH .
CURRENT OPINION IN GENETICS & DEVELOPMENT, 1994, 4 (03) :466-471
[16]  
Cooper MJ, 1996, SEMIN ONCOL, V23, P172
[17]   GENE-TRANSFER TO THE THYMUS - A MEANS OF ABROGATING THE IMMUNE-RESPONSE TO RECOMBINANT ADENOVIRUS [J].
DEMATTEO, RP ;
RAPER, SE ;
AHN, M ;
FISHER, KJ ;
BURKE, C ;
RADU, A ;
WIDERA, G ;
CLAYTOR, BR ;
BARKER, CF ;
MARKMANN, JF .
ANNALS OF SURGERY, 1995, 222 (03) :229-242
[18]   Elimination of the carboxy-terminal sequences of parathyroid hormone-related protein 1-173 increases production and secretion of the truncated forms [J].
Ditmer, LS ;
Burton, DW ;
Deftos, LJ .
ENDOCRINOLOGY, 1996, 137 (05) :1608-1617
[19]   Resurfacing of articular cartilage explants with genetically-modified human chondrocytes in vitro [J].
Doherty, PJ ;
Zhang, HW ;
Tremblay, L ;
Manolopoulos, V ;
Marshall, KW .
OSTEOARTHRITIS AND CARTILAGE, 1998, 6 (03) :153-159
[20]   Chondrogenic phenotype of perichondrium-derived chondroprogenitor cells is influenced by transforming growth factor-beta 1 [J].
Dounchis, JS ;
Goomer, RS ;
Harwood, FL ;
Khatod, M ;
Coutts, RD ;
Amiel, D .
JOURNAL OF ORTHOPAEDIC RESEARCH, 1997, 15 (06) :803-807