Research of enhanced green fluorescent protein gene transfer with ultrasound-mediated microbubble destruction in bone defects_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Shiwei ¹ , XIE Xiaoli ² , YANG Xiaodong ¹ , LIU Lijun ¹ ,  TANG Xueyang ¹

1. Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;
2. Department of Pediatric Surgery, Guangzhou Women and Children’s Medical Center, Guangzhou Guangdong, 510623, P.R.China;

Corresponding?author：

TANG Xueyang, Email: tangxueyang100@163.com

Keywords：

Bone defect; ultrasound-mediated microbubble destruction; gene transfer; enhanced green fluorescent protein; rabbit

DOI：

10.7507/1002-1892.201611059

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Methods Thirty 3-month-old New Zealand rabbits (2.5-3.0 kg in weight) were randomly divided into 5 groups (n=6) and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected (0.3 mL/kg) and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm², and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively (in 1, 2, 3, 4, and 5 minutes groups respectively). The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. Results The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance (A) value showed significant differences when compared 1 minute and 2 minutes groups with other groups (P<0.05), but no significant difference was found between 3, 4, and 5 minutes groups (P>0.05). Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. Conclusion EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm², and duty ratio of 20% for 2 minutes in bone defects of rabbits.

Citation： LI Shiwei, XIE Xiaoli, YANG Xiaodong, LIU Lijun, TANG Xueyang. Research of enhanced green fluorescent protein gene transfer with ultrasound-mediated microbubble destruction in bone defects. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(4): 437-442. doi: 10.7507/1002-1892.201611059 Copy

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12.	Song S, Shen Z, Chen L,et al. Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver. Gene Ther, 2011, 18(10): 1006-1014.
13.	Fujii H, Li SH, Wu J,et al. Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair. Eur Heart J, 2011, 32(16): 2075-2084.
14.	Zhang Y, Ye C, Wang G,et al. Kidney-targeted transplantation of mesenchymal stem cells by ultrasound-targeted microbubble destruction promotes kidney repair in diabetic nephropathy rats. Biomed Res Int, 2013, 2013: 526367.
15.	Tinkov S, Bekeredjian R, Winter G,et al. Microbubbles as ultrasound triggered drug carriers. J Pharm Sci, 2009, 98(6): 1935-1961.
16.	陳智毅, 謝明星, 王新房, 等. 治療性超聲介導體外基因轉染的參數優化. 中國醫學影像技術, 2008, 24(10): 1511-1514.
17.	Liang HD, Lu QL, Xue SA,et al. Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells. Ultrasound Med Biol, 2004, 30(11): 1523-1529.
18.	Koch S, Pohl P, Cobet U,et al. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound Med Biol, 2000, 26(5): 897-903.
19.	Delalande A, Bouakaz A, Renault G,et al. Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype. J Control Release, 2011, 156(2): 223-230.
20.	Zarnitsyn VG, Prausnitz MR. Physical parameters influencing optimization of ultrasound-mediated DNA transfection. Ultrasound Med Biol, 2004, 30(4): 527-538.
21.	Wang X, Liang HD, Dong B,et al. Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents. Radiology, 2005, 237(1): 224-229.
22.	馬釗, 胡向東, 費琦, 等. 超聲微泡介導基因治療在骨科領域的應用進展. 臨床和實驗醫學雜志, 2014, 13(6): 505-508.

1. Wiese A, Pape HC. Bone defects caused by high-energy injuries, bone loss, infected nonunions, and nonunions. Orthop Clin North Am, 2010, 41(1): 1-4.
2. Karger C, Kishi T, Schneider L,et al. Treatment of posttraumatic bone defects by the induced membrane technique. Orthop Traumatol Surg Res, 2012, 98(1): 97-102.
3. Wang Y, Van Manh N, Wang H,et al. Synergistic intra-fibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to promote the regeneration of bone defects. Int J Nanomedicine, 2016, 11: 2053-2067.
4. Kolambkar YM, Dupont KM, Boerckel JD,et al. An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. Biomaterials, 2011, 32(1): 65-74.
5. Van der Stok J, Van der Jagt OP, Amin Yavari S,et al. Selective laser melting-produced porous titanium scaffolds regenerate bone in critical size cortical bone defects. J Orthop Res, 2013, 31(5): 792-799.
6. 殷渠東, 孫振中, 顧三軍, 等. 骨搬運與骨短縮-延長治療脛骨骨缺損合并軟組織缺損的療效比較. 中國修復重建外科雜志, 2014, 28(7): 818-822.
7. Long J, Li P, Du H,et al. Effects of bone morphogenetic protein 2 gene therapy on new bone formation during mandibular distraction osteogenesis at rapid rate in rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2011, 112(1): 50-57.
8. Gugala Z, Olmsted-Davis EA, Gannon FH,et al. Osteoinduction byex vivo adenovirus-mediated BMP2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.
9. Qiu L, Zhang L, Wang L,et al. Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendonsin vivo. Gene Ther, 2012, 19(7): 703-710.
10. Osawa K, Okubo Y, Nakao K,et al. Osteoinduction by micro-bubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2. J Med, 2009, 11(7): 633-641.
11. Chen ZY, Yang F, Lin Y,et al. New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery. Curr Gene Ther, 2013, 13(4): 250-274.
12. Song S, Shen Z, Chen L,et al. Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver. Gene Ther, 2011, 18(10): 1006-1014.
13. Fujii H, Li SH, Wu J,et al. Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair. Eur Heart J, 2011, 32(16): 2075-2084.
14. Zhang Y, Ye C, Wang G,et al. Kidney-targeted transplantation of mesenchymal stem cells by ultrasound-targeted microbubble destruction promotes kidney repair in diabetic nephropathy rats. Biomed Res Int, 2013, 2013: 526367.
15. Tinkov S, Bekeredjian R, Winter G,et al. Microbubbles as ultrasound triggered drug carriers. J Pharm Sci, 2009, 98(6): 1935-1961.
16. 陳智毅, 謝明星, 王新房, 等. 治療性超聲介導體外基因轉染的參數優化. 中國醫學影像技術, 2008, 24(10): 1511-1514.
17. Liang HD, Lu QL, Xue SA,et al. Optimisation of ultrasound-mediated gene transfer (sonoporation) in skeletal muscle cells. Ultrasound Med Biol, 2004, 30(11): 1523-1529.
18. Koch S, Pohl P, Cobet U,et al. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound Med Biol, 2000, 26(5): 897-903.
19. Delalande A, Bouakaz A, Renault G,et al. Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype. J Control Release, 2011, 156(2): 223-230.
20. Zarnitsyn VG, Prausnitz MR. Physical parameters influencing optimization of ultrasound-mediated DNA transfection. Ultrasound Med Biol, 2004, 30(4): 527-538.
21. Wang X, Liang HD, Dong B,et al. Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents. Radiology, 2005, 237(1): 224-229.
22. 馬釗, 胡向東, 費琦, 等. 超聲微泡介導基因治療在骨科領域的應用進展. 臨床和實驗醫學雜志, 2014, 13(6): 505-508.

Chinese Journal of Reparative and Reconstructive Surgery

Research of enhanced green fluorescent protein gene transfer with ultrasound-mediated microbubble destruction in bone defects

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