Ultrasound-responsive gene-activated matrices (GAMs) for osteogenic gene therapy using matrix-assisted sonoporation (MAS)

N Nomikou, GA Feichtinger, S Saha, S Nuernberger, P Heimel, H Redl, AP McHale

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here we describe an advanced ultrasound-responsive GAM containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 co-expression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immuno-histochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography (µCT) and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies.
LanguageEnglish
Pagese250-260
Number of pages10
JournalJournal of Tissue Engineering and Regenerative Medicine
Volume12
Issue number1
Early online date1 Jun 2017
DOIs
Publication statusPublished - 1 Jan 2018

Fingerprint

Gene therapy
Genetic Therapy
Genes
Ultrasonics
Bone
Bone Morphogenetic Protein 7
Gene transfer
Bone and Bones
Microbubbles
X-Ray Microtomography
Tissue regeneration
Bone Morphogenetic Proteins
Osteocalcin
Phosphatases
Fibrin
Transgenes
Collagen
Tomography
Alkaline Phosphatase
Regeneration

Keywords

  • Osteogenesis
  • gene
  • matrix
  • sonoporation
  • ultrasound
  • regeneration

Cite this

@article{cff02f15ded5498ab671498dd13be00a,
title = "Ultrasound-responsive gene-activated matrices (GAMs) for osteogenic gene therapy using matrix-assisted sonoporation (MAS)",
abstract = "Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here we describe an advanced ultrasound-responsive GAM containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 co-expression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immuno-histochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography (µCT) and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies.",
keywords = "Osteogenesis, gene, matrix, sonoporation, ultrasound, regeneration",
author = "N Nomikou and GA Feichtinger and S Saha and S Nuernberger and P Heimel and H Redl and AP McHale",
note = "Reference text: Alhadlaq, J.M. 2004, Mesenchymal Stem Cells: Isolation and Therapeutics. Stem Cells and Development, 2004. 13: 436-448. Balmayor ER, van Griensven M. 2015, Gene therapy for bone engineering. Front Bioeng Biotechnol, 3:9, doi: 10.3389/fbioe.2015.00009 Bonadio J. 2000, Tissue engineering via local gene delivery: Update and future prospects for enhancing the technology. Adv Drug Deliv Rev, 44:185-194. Entezari V, Vartanians V, Zurakowski D, Patel N, Fajardo RJ, Muller R, Snyder BD, Nazarian A. 2012, Further improvements on the factors affecting bone mineral density measured by quantitative micro-computed tomography. Bone 50:611-618. Escoffre JM, Zeghimi A, Novell A, Bouakaz A. 2013, In-vivo gene delivery by sonoporation: recent progress and prospects. Current Gene Ther, 13:2-14. Evans CH, Huard J. 2015, Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol, 11:234-242. Feichtinger GA, Hacobian A, Hofmann AT, Wassermann K, Zimmermann A, van Griensven M, Redl H. 2014a, Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy. Eur Cell Mater, 27:166-184; discussion 184. Feichtinger GA, Hofmann AT, Slezak P, Schuetzenberger S, Kaipel M, Schwartz E, Neef A, Nomikou N, Nau T, van Griensven M, McHale AP, Redl H. 2014. Sonoporation increases therapeutic efficacy of inducible and constitutive BMP2/7 in vivo gene delivery. Hum Gene Ther Methods, 25:57-71. Gafni Y, G.T., Liebergal GT, Pelled G, Gazit Z, Gazit D. 2004, Stem cells as vehicles for orthopaedic gene therapy. Gene Therapy. 11: 417-426. Gonzalez AM, Berry M, Greenlees L, Logan A, Baird A. 2006, Matrix-mediated gene transfer to brain cortex and dorsal root ganglion neurones by retrograde axonal transport after dorsal column lesion. J Gene Med, 8: 901-909. Hustedt JW, Blizzard DJ. 2014, The controversy surrounding bone morphogenetic proteins in the spine: a review of current research. Yale J Biol Med, 87:549-561. Jafari M, Soltani M, Naahidi S, Karunaratne DN, Chen P. 2012, Nonviral approach for targeted nucleic acid delivery. Curr Med Chem, 19:197-208. Javed A, Chen H, Ghori FY. 2010, Genetic and transcriptional control of bone formation. Oral Maxillofac Surg Clin North Am, 22:283-293. Kawai M, Bessho K, Maruyama H, Miyazaki J, Yamamoto T. 2006, Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression. BMC Musculoskelet Disord, 7:62, doi:10.1186/1471-2474-7-62 Kayabasi GK, Aydin RS, Gumusderelioglu M. 2013, In vitro chondrogenesis by BMP6 gene therapy. J Biomed Mater Res A, 101:1353-61. Kimelman-Bleich N, Pelled G, Zilberman Y, Kallai I, Mizrahi O, Tawackoli W, Gazit Z, Gazit D. 2011, Mol Ther, 19, 53-59. Lammertink BH, Bos C, Deckers R, Storm G, Moonen CT, Escoffre JM. 2015, Sonochemotherapy: From bench to bedside. Front Pharmacol, 6:138, doi: 10.3389/fphar.2015.00138 Li G, Peng H, Corsi K, Usas A, Olshanski A, Huard J. 2005, Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation. J Bone Miner Res, 20:1611-1623. Lu CH, Chang YH, Lin SY, Li KC, Hu YC. 2013, Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv, 31:1695-706. McEwan C, Fowley C, Nomikou N, McCaughan B, McHale AP, Callan JF. 2014, Polymeric microbubbles as delivery vehicles for sensitizers in sonodynamic therapy. Langmuir, 30:14926-14930. Michlits W, Mittermayr R, Schafer R, Redl H, Aharinejad S. 2007, Fibrin-embedded administration of VEGF plasmid enhances skin flap survival. Wound Repair Regen, 15:360-367. Nomikou N, Feichtinger GA, Redl H, McHale AP. 2016, Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration. J Tissue Eng Regen Med, 10:29-39. Nomikou N, Tiwari P, Trehan T, Gulati K, McHale AP. 2012, Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene delivery in vitro and in vivo. Acta Biomater, 8:1273-1280. Osawa K, Okubo Y, Nakao K, Koyama N, Bessho K. 2009, Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2. J Gene Med, 11:633-641. Osawa K, Okubo Y, Nakao K, Koyama N, Bessho K. 2010, Osteoinduction by repeat plasmid injection of human bone morphogenetic protein-2. J Gene Med, 12:937-44. Pascher A, Palmer GD, Steinert A, Oligino T, Gouze E, Gouze JN, Betz O, Spector M, Robbins PD, Evans CH, Ghivizzani SC. 2004, Gene delivery to cartilage defects using coagulated bone marrow aspirate. Gene Ther, 11:133-141. Peterson CY, Shaterian A, Borboa AK, Gonzalez AM, Potenza BM, Coimbra R, Eliceiri BP, Baird A. 2009, The noninvasive, quantitative, in vivo assessment of adenoviral-mediated gene delivery in skin wound biomaterials. Biomaterials, 30:6788-6793. Ramamoorth M, Narvekar A. 2015, Non viral vectors in gene therapy- an overview. J Clin Diagn Res, 9:GE01-6. Schillinger U, Wexel G, Hacker C, Kullmer M, Koch C, Gerg M, Vogt S, Ueblacker P, Tischer T, Hensler D, Wilisch J, Ainer J, Walch A, Stemberger A, Plank C, 2008, A fibrin glue composition as carrier for nucleic acid vectors. Pharm Res, 25:2946-2962. Tierney EG, Duffy GP, Cryan SA, Curtin CM, O'Brien FJ. 2013, Non-viral gene-activated matrices: next generation constructs for bone repair. Organogenesis, 9:22-8. Wang W, Li W, Ma N, Steinhoff G. 2013, Non-viral gene delivery methods. Curr Pharm Biotechnol, 14:46-60. Wang X, Helary C, CoradinT. 2015, Local and sustained gene delivery in silica-collagen nanocomposites. ACS Appl. Mater. Interfaces, 7:2503-2511. Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. 2014, Non-viral vectors for gene-based therapy. Nat Rev Genet, 15:541-55. Zhang, W., C. Zhu, Y. Wu, D. Ye, S. Wang, D. Zou, X. Zhang, D.L. Kaplan, and X. Jiang. 2014. VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation. Eur Cell Mater. 27:1-11.",
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Ultrasound-responsive gene-activated matrices (GAMs) for osteogenic gene therapy using matrix-assisted sonoporation (MAS). / Nomikou, N; Feichtinger, GA; Saha, S; Nuernberger, S; Heimel, P; Redl, H; McHale, AP.

In: Journal of Tissue Engineering and Regenerative Medicine, Vol. 12, No. 1, 01.01.2018, p. e250-260.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ultrasound-responsive gene-activated matrices (GAMs) for osteogenic gene therapy using matrix-assisted sonoporation (MAS)

AU - Nomikou, N

AU - Feichtinger, GA

AU - Saha, S

AU - Nuernberger, S

AU - Heimel, P

AU - Redl, H

AU - McHale, AP

N1 - Reference text: Alhadlaq, J.M. 2004, Mesenchymal Stem Cells: Isolation and Therapeutics. Stem Cells and Development, 2004. 13: 436-448. Balmayor ER, van Griensven M. 2015, Gene therapy for bone engineering. Front Bioeng Biotechnol, 3:9, doi: 10.3389/fbioe.2015.00009 Bonadio J. 2000, Tissue engineering via local gene delivery: Update and future prospects for enhancing the technology. Adv Drug Deliv Rev, 44:185-194. Entezari V, Vartanians V, Zurakowski D, Patel N, Fajardo RJ, Muller R, Snyder BD, Nazarian A. 2012, Further improvements on the factors affecting bone mineral density measured by quantitative micro-computed tomography. Bone 50:611-618. Escoffre JM, Zeghimi A, Novell A, Bouakaz A. 2013, In-vivo gene delivery by sonoporation: recent progress and prospects. Current Gene Ther, 13:2-14. Evans CH, Huard J. 2015, Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol, 11:234-242. Feichtinger GA, Hacobian A, Hofmann AT, Wassermann K, Zimmermann A, van Griensven M, Redl H. 2014a, Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy. Eur Cell Mater, 27:166-184; discussion 184. Feichtinger GA, Hofmann AT, Slezak P, Schuetzenberger S, Kaipel M, Schwartz E, Neef A, Nomikou N, Nau T, van Griensven M, McHale AP, Redl H. 2014. Sonoporation increases therapeutic efficacy of inducible and constitutive BMP2/7 in vivo gene delivery. Hum Gene Ther Methods, 25:57-71. Gafni Y, G.T., Liebergal GT, Pelled G, Gazit Z, Gazit D. 2004, Stem cells as vehicles for orthopaedic gene therapy. Gene Therapy. 11: 417-426. Gonzalez AM, Berry M, Greenlees L, Logan A, Baird A. 2006, Matrix-mediated gene transfer to brain cortex and dorsal root ganglion neurones by retrograde axonal transport after dorsal column lesion. J Gene Med, 8: 901-909. Hustedt JW, Blizzard DJ. 2014, The controversy surrounding bone morphogenetic proteins in the spine: a review of current research. Yale J Biol Med, 87:549-561. Jafari M, Soltani M, Naahidi S, Karunaratne DN, Chen P. 2012, Nonviral approach for targeted nucleic acid delivery. Curr Med Chem, 19:197-208. Javed A, Chen H, Ghori FY. 2010, Genetic and transcriptional control of bone formation. Oral Maxillofac Surg Clin North Am, 22:283-293. Kawai M, Bessho K, Maruyama H, Miyazaki J, Yamamoto T. 2006, Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression. BMC Musculoskelet Disord, 7:62, doi:10.1186/1471-2474-7-62 Kayabasi GK, Aydin RS, Gumusderelioglu M. 2013, In vitro chondrogenesis by BMP6 gene therapy. J Biomed Mater Res A, 101:1353-61. Kimelman-Bleich N, Pelled G, Zilberman Y, Kallai I, Mizrahi O, Tawackoli W, Gazit Z, Gazit D. 2011, Mol Ther, 19, 53-59. Lammertink BH, Bos C, Deckers R, Storm G, Moonen CT, Escoffre JM. 2015, Sonochemotherapy: From bench to bedside. Front Pharmacol, 6:138, doi: 10.3389/fphar.2015.00138 Li G, Peng H, Corsi K, Usas A, Olshanski A, Huard J. 2005, Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation. J Bone Miner Res, 20:1611-1623. Lu CH, Chang YH, Lin SY, Li KC, Hu YC. 2013, Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv, 31:1695-706. McEwan C, Fowley C, Nomikou N, McCaughan B, McHale AP, Callan JF. 2014, Polymeric microbubbles as delivery vehicles for sensitizers in sonodynamic therapy. Langmuir, 30:14926-14930. Michlits W, Mittermayr R, Schafer R, Redl H, Aharinejad S. 2007, Fibrin-embedded administration of VEGF plasmid enhances skin flap survival. Wound Repair Regen, 15:360-367. Nomikou N, Feichtinger GA, Redl H, McHale AP. 2016, Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration. J Tissue Eng Regen Med, 10:29-39. Nomikou N, Tiwari P, Trehan T, Gulati K, McHale AP. 2012, Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene delivery in vitro and in vivo. Acta Biomater, 8:1273-1280. Osawa K, Okubo Y, Nakao K, Koyama N, Bessho K. 2009, Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2. J Gene Med, 11:633-641. Osawa K, Okubo Y, Nakao K, Koyama N, Bessho K. 2010, Osteoinduction by repeat plasmid injection of human bone morphogenetic protein-2. J Gene Med, 12:937-44. Pascher A, Palmer GD, Steinert A, Oligino T, Gouze E, Gouze JN, Betz O, Spector M, Robbins PD, Evans CH, Ghivizzani SC. 2004, Gene delivery to cartilage defects using coagulated bone marrow aspirate. Gene Ther, 11:133-141. Peterson CY, Shaterian A, Borboa AK, Gonzalez AM, Potenza BM, Coimbra R, Eliceiri BP, Baird A. 2009, The noninvasive, quantitative, in vivo assessment of adenoviral-mediated gene delivery in skin wound biomaterials. Biomaterials, 30:6788-6793. Ramamoorth M, Narvekar A. 2015, Non viral vectors in gene therapy- an overview. J Clin Diagn Res, 9:GE01-6. Schillinger U, Wexel G, Hacker C, Kullmer M, Koch C, Gerg M, Vogt S, Ueblacker P, Tischer T, Hensler D, Wilisch J, Ainer J, Walch A, Stemberger A, Plank C, 2008, A fibrin glue composition as carrier for nucleic acid vectors. Pharm Res, 25:2946-2962. Tierney EG, Duffy GP, Cryan SA, Curtin CM, O'Brien FJ. 2013, Non-viral gene-activated matrices: next generation constructs for bone repair. Organogenesis, 9:22-8. Wang W, Li W, Ma N, Steinhoff G. 2013, Non-viral gene delivery methods. Curr Pharm Biotechnol, 14:46-60. Wang X, Helary C, CoradinT. 2015, Local and sustained gene delivery in silica-collagen nanocomposites. ACS Appl. Mater. Interfaces, 7:2503-2511. Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. 2014, Non-viral vectors for gene-based therapy. Nat Rev Genet, 15:541-55. Zhang, W., C. Zhu, Y. Wu, D. Ye, S. Wang, D. Zou, X. Zhang, D.L. Kaplan, and X. Jiang. 2014. VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation. Eur Cell Mater. 27:1-11.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here we describe an advanced ultrasound-responsive GAM containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 co-expression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immuno-histochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography (µCT) and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies.

AB - Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here we describe an advanced ultrasound-responsive GAM containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 co-expression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immuno-histochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography (µCT) and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies.

KW - Osteogenesis

KW - gene

KW - matrix

KW - sonoporation

KW - ultrasound

KW - regeneration

U2 - 10.1002/term.2406

DO - 10.1002/term.2406

M3 - Article

VL - 12

SP - e250-260

JO - Journal of Tissue Engineering and Regenerative Medicine

T2 - Journal of Tissue Engineering and Regenerative Medicine

JF - Journal of Tissue Engineering and Regenerative Medicine

SN - 1932-7005

IS - 1

ER -