A Fibrin Glue Composition as Carrier for Nucleic Acid Vectors
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Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration.
Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL® and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model.
The optimized glue is based on the fibrinogen component of TISSUCOL®, a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group).
COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.
KEY WORDSgene-activated matrix fibrinogen gene therapy gene transfer tissue adhesive tissue engineering
This work was supported by the Deutsche Forschungsgemeinschaft (Pl 281/1) and Baxter AG, Vienna, Austria, and the German Federal Ministry of Education and Research (grant 0312019A).
- 7.G. Schmidmaier, B. Wildemann, T. Gabelein, J. Heeger, F. Kandziora, N. P. Haas, and M. Raschke. Synergistic effect of IGF-I and TGF-beta1 on fracture healing in rats: single versus combined application of IGF-I and TGF-beta1. Acta Orthop. Scand. 74:604–610 (2003). doi: 10.1080/00016470310018036.PubMedCrossRefGoogle Scholar
- 8.H. Gollwitzer, P. Thomas, P. Diehl, E. Steinhauser, B. Summer, S. Barnstorf, L. Gerdesmeyer, W. Mittelmeier, and A. Stemberger. Biomechanical and allergological characteristics of a biodegradable poly(D,L-lactic acid) coating for orthopaedic implants. J. Orthop. Res. 23:802–809 (2005). doi: 10.1016/j.orthres.2005.02.003.PubMedCrossRefGoogle Scholar
- 17.C. Andree, M. Voigt, A. Wenger, T. Erichsen, K. Bittner, D. Schaefer, K. J. Walgenbach, J. Borges, R. E. Horch, E. Eriksson, and G. B. Stark. Plasmid gene delivery to human keratinocytes through a fibrin-mediated transfection system. Tissue Eng. 7:757–766 (2001). doi: 10.1089/107632701753337708.PubMedCrossRefGoogle Scholar
- 19.K. L. Christman, Q. Fang, M. S. Yee, K. R. Johnson, R. E. Sievers, and R. J. Lee. Enhanced neovasculature formation in ischemic myocardium following delivery of pleiotrophin plasmid in a biopolymer. Biomaterials. 26:1139–1144 (2005). doi: 10.1016/j.biomaterials.2004.04.025.PubMedCrossRefGoogle Scholar
- 20.D. Trentin, H. Hall, S. Wechsler, and J. A. Hubbell. Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis. Proc. Natl. Acad. Sci. USA. 103:2506–2511 (2006). doi: 10.1073/pnas.0505964102.PubMedCrossRefGoogle Scholar
- 22.A. Breen, P. Dockery, T. O’Brien, and A. Pandit. Fibrin scaffold promotes adenoviral gene transfer and controlled vector delivery. J Biomed Mater Res A. In press (2008).Google Scholar
- 26.M. J. Escamez, M. Carretero, M. Garcia, L. Martinez-Santamaria, I. Mirones, B. Duarte, A. Holguin, E. Garcia, V. Garcia, A. Meana, J. L. Jorcano, F. Larcher, and M. Del Rio. Assessment of optimal virus-mediated growth factor gene delivery for human cutaneous wound healing enhancement. J. Invest. Dermatol. 128:1565–1575 (2008). doi: 10.1038/sj.jid.5701217.PubMedCrossRefGoogle Scholar
- 28.F. Teraishi, T. Umeoka, T. Saito, T. Tsukagoshi, N. Tanaka, and T. Fujiwara. A novel method for gene delivery and expression in esophageal epithelium with fibrin glues containing replication-deficient adenovirus vector. Surg. Endosc. 17:1845–1848 (2003). doi: 10.1007/s00464-003-8146-5.PubMedCrossRefGoogle Scholar
- 33.M. Arlt, C. Kopitz, C. Pennington, K. L. Watson, H. W. Krell, W. Bode, B. Gansbacher, R. Khokha, D. R. Edwards, and A. Kruger. Increase in gelatinase-specificity of matrix metalloproteinase inhibitors correlates with antimetastatic efficacy in a T-cell lymphoma model. Cancer Res. 62:5543–5550 (2002).PubMedGoogle Scholar
- 34.P. Mainil-Varlet, T. Aigner, M. Brittberg, P. Bullough, A. Hollander, E. Hunziker, R. Kandel, S. Nehrer, K. Pritzker, S. Roberts, and E. Stauffer. Histological assessment of cartilage repair: a report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J. Bone Joint Surg. Am. 85-A(Suppl 2):45–57 (2003).PubMedGoogle Scholar
- 39.J. Fang, Y. Y. Zhu, E. Smiley, J. Bonadio, J. P. Rouleau, S. A. Goldstein, L. K. McCauley, B. L. Davidson, and B. J. Roessler. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes. Proc. Natl. Acad. Sci. U.S.A. 93:5753–5758 (1996). doi: 10.1073/pnas.93.12.5753.PubMedCrossRefGoogle Scholar
- 40.C. Perka, R. S. Spitzer, K. Lindenhayn, M. Sittinger, and O. Schultz. Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants. J. Biomed. Mater. Res. 49:305–311 (2000)doi: 10.1002/(SICI)1097-4636(20000305)49:3<305::AID-JBM2>3.0.CO;2-9.PubMedCrossRefGoogle Scholar
- 46.P. Erbacher, T. Bettinger, P. Belguise-Valladier, S. Zou, J. L. Coll, J. P. Behr, and J. S. Remy. Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI). J. Gene Med. 1:210–222 (1999). doi: 10.1002/(SICI)1521-2254(199905/06)1:3<210::AID-JGM30>3.0.CO;2-U.PubMedCrossRefGoogle Scholar
- 49.S. Vogt, P. Ueblacker, C. Geis, B. Wagner, G. Wexel, T. Tischer, A. Kruger, C. Plank, M. Anton, V. Martinek, A. B. Imhoff, and B. Gansbacher. Efficient and stable gene transfer of growth factors into chondrogenic cells and primary articular chondrocytes using a VSV.G pseudotyped retroviral vector. Biomaterials. 29:1242–1249 (2008). doi: 10.1016/j.biomaterials.2007.11.013.PubMedCrossRefGoogle Scholar
- 50.K. Gelse, Q. J. Jiang, T. Aigner, T. Ritter, K. Wagner, E. Poschl, K. von der Mark, and H. Schneider. Fibroblast-mediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer. Arthritis Rheum. 44:1943–1953 (2001)doi: 10.1002/1529-0131(200108)44:8<1943::AID-ART332>3.0.CO;2-Z.PubMedCrossRefGoogle Scholar
- 51.P. Ueblacker, B. Wagner, S. Vogt, G. Salzmann, G. Wexel, A. Kruger, C. Plank, T. Brill, K. Specht, T. Hennig, U. Schillinger, A. B. Imhoff, V. Martinek, and B. Gansbacher. In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects. Biomaterials. 28:4480–4487 (2007). doi: 10.1016/j.biomaterials.2007.06.027.PubMedCrossRefGoogle Scholar
- 52.S. C. Hyde, I. A. Pringle, S. Abdullah, A. E. Lawton, L. A. Davies, A. Varathalingam, G. Nunez-Alonso, A. M. Green, R. P. Bazzani, S. G. Sumner-Jones, M. Chan, H. Li, N. S. Yew, S. H. Cheng, A. C. Boyd, J. C. Davies, U. Griesenbach, D. J. Porteous, D. N. Sheppard, F. M. Munkonge, E. W. Alton, and D. R. Gill. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat. Biotechnol. 26:549–551 (2008). doi: 10.1038/nbt1399.PubMedCrossRefGoogle Scholar