Construction of injectable tissue engineered adipose tissue with fibrin glue scaffold and human adipose-derived stem cells transfected by lentivirus vector expressing hepatocyte growth factor_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHU Yuanzheng ,  YI Yangyan , YANG Shuifa , ZHANG Jing , WU Shu , WANG Zhaohui

Department of Plastic Surgery, Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China;

Corresponding?author：

YI Yangyan, Email: yyy0218@126.com

Keywords：

Tissue engineered adipose; human adipose-derived stem cells; fibrin glue; hepatocyte growth factor; lentivirus; gene transfection; nude mouse

DOI：

10.7507/1002-1892.201704094

Video：

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ObjectiveTo discuss the possibility of constructing injectable tissue engineered adipose tissue, and to provide a new approach for repairing soft tissue defects.MethodsHuman adipose-derived stem cells (hADSCs) were extracted from the lipid part of human liposuction aspirate by enzymatic digestion and identified by morphological observation, flow cytometry, and adipogenic induction. The hADSCs underwent transfection by lentivirus vector expressing hepatocyte growth factor and green fluorescent protein (HGF-GFP-LVs) of different multiplicity of infection (MOI, 10, 30, 50, and 100), the transfection efficiency was calculated to determine the optimum MOI. The hADSCs transfected by HGF-GFP-LVs of optimal MOI and being adipogenic inducted were combined with injectable fibrin glue scaffold, and were injected subcutaneously into the right side of the low back of 10 T-cell deficiency BALB/c female nude mice (transfected group); non-HGF-GFP-LVs transfected hADSCs (being adipogenic inducted) combined with injectable fibrin glue scaffold were injected subcutaneously into the left side of the low back (untransfected group); and injectable fibrin glue scaffold were injected subcutaneously into the middle part of the neck (blank control group); 0.4 mL at each point. Twelve weeks later the mice were killed and the implants were taken out. Gross observation, wet weight measurement, HE staining, GFP fluorescence labeling, and immunofluorescence staining were performed to assess the in vivo adipogenic ability of the seed cells and the neovascularization of the grafts.ResultsThe cultured cells were identified as hADSCs. Poor transfection efficiency was observed in MOI of 10 and 30, the transfection efficiency of MOI of 50 and 100 was more than 80%, so the optimum MOI was 50. Adipose tissue-like new-born tissues were found in the injection sites of the transfected and untransfected groups after 12 weeks of injection, and no new-born tissues was found in the blank control group. The wet-weight of new-born tissue in the transfected group [(32.30±4.06) mg] was significantly heavier than that of the untransfected group [(25.27±3.94) mg] (t=3.929, P=0.001). The mature adipose cells in the transfected group [(126.93±5.36) cells/field] were significantly more than that in the untransfected group [(71.36±4.52) cells/field] (t=30.700, P=0.000). Under fluorescence microscopy, some of the single cell adipocytes showed a network of green fluorescence, indicating the presence of GFP labeled exogenous hADSCs in the tissue. The vascular density of new-born tissue of the transfected group [(16.37±2.76)/field] was significantly higher than that of the untransfected group [(9.13±1.68)/field] (t=8.678, P=0.000).ConclusionThe hADSCs extracted from the lipid part after liposuction can be used as seed cells. After HGF-GFP-LVs transfection and adipose induction, the hADSCs combined with injectable fibrin glue scaffold can construct mature adipose tissue in vivo, which may stimulate angiogenesis, and improve retention rate of new-born tissue.

Citation： ZHU Yuanzheng, YI Yangyan, YANG Shuifa, ZHANG Jing, WU Shu, WANG Zhaohui. Construction of injectable tissue engineered adipose tissue with fibrin glue scaffold and human adipose-derived stem cells transfected by lentivirus vector expressing hepatocyte growth factor. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(9): 1111-1118. doi: 10.7507/1002-1892.201704094 Copy

1.	Chang Q, Lu F. A novel strategy for creating a large amount of engineered fat tissue with an axial vascular pedicle and a prefabricated scaffold. Med Hypotheses, 2012, 79(2): 267-270.
2.	陽水發, 易陽艷. 脂肪干細胞構建可注射型組織工程脂肪的研究進展. 中國修復重建外科雜志, 2015, 29(2): 245-249.
3.	陽水發, 易陽艷, 黃艷, 等. 人脂肪干細胞與不同濃度纖維蛋白膠的相容性研究. 重慶醫學, 2017, 46(2): 165-168.
4.	Murphy KC, Fang SY, Leach JK. Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell Tissue Res, 2014, 357(1): 91-99.
5.	陽水發, 易陽艷, 黃艷, 等. 腺病毒介導 GFP 基因標記人脂肪來源干細胞可行性研究. 中國美容整形外科雜志, 2016, 27(8): 495-498.
6.	Cecchi F, Rabe DC, Bottaro DP. Targeting the HGF/Met signaling pathway in cancer therapy. Expert Opin Ther Targets, 2012, 16(6): 553-572.
7.	Nakamura T, Mizuno S. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci, 2010, 86(6): 588-610.
8.	焦志勇, 羅敏, 李盛, 等. 肝細胞生長因子對人羊水干細胞遷移和黏附能力的影響. 中華細胞與干細胞雜志, 2013, 3(3): 139-143.
9.	Liu J, Pan G, Liang T, et al. HGF/c-Met signaling mediated mesenchymal stem cell-induced liver recovery in intestinal ischemia reperfusion model. Int J Med Sci, 2014, 11(6): 626-633.
10.	Abd EN, Abdulrahman M, Helal M, et al. Regenerative capacity of allogenic gingival margin-derived stem cells with fibrin glue on albino rats’partially dissected submandibular salivary glands. Arch Oral Biol, 2017, 82: 302-309.
11.	Kim YS, Sung CH, Chung SH, et al. Does an Injection of Adipose-Derived Mesenchymal Stem Cells Loaded in Fibrin Glue Influence Rotator Cuff Repair Outcomes? A Clinical and Magnetic Resonance Imaging Study. Am J Sports Med, 2017, 45(9): 2010-2018.
12.	Hao C, Wang Y, Shao L, et al. Local Injection of Bone Mesenchymal Stem Cells and Fibrin Glue Promotes the Repair of Bone Atrophic Nonunion In Vivo. Adv Ther, 2016, 33(5): 824-833.
13.	Liu Z, Zhu Y, Zhu H, et al. Enhancement of posterolateral lumbar spine fusion using recombinant human bone morphogenetic protein-2 and mesenchymal stem cells delivered in fibrin glue. J Biomater Appl, 2016, 31(4): 477-487.
14.	Mehanna RA, Nabil I, Attia N, et al. The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats. Biomed Res Int, 2015, 2015: 846062.
15.	Yang Y, Zhang W, Li Y, et al. Scalded skin of rat treated by using fibrin glue combined with allogeneic bone marrow mesenchymal stem cells. Ann Dermatol, 2014, 26(3): 289-295.
16.	Wen Q, Zhou C, Luo W, et al. Pro-osteogenic effects of fibrin glue in treatment of avascular necrosis of the femoral head in vivo by hepatocyte growth factor-transgenic mesenchymal stem cells. J Transl Med, 2014, 12: 114.
17.	Mcduffee LA, Esparza GB, Nino-Fong R, et al. Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold. Cell Tissue Res, 2014, 355(2): 327-335.
18.	Wang K, Guan Y, Liu Y, et al. Fibrin glue with autogenic bone marrow mesenchymal stem cells for urethral injury repair in rabbit model. Tissue Eng Part A, 2012, 18(23-24): 2507-2517.
19.	Ferris D, Frisbie D, Kisiday J, et al. In vivo healing of meniscal lacerations using bone marrow-derived mesenchymal stem cells and fibrin glue. Stem Cells Int, 2012, 2012: 691605.
20.	Zhang L, Wang P, Mei S, et al. In vivo alveolar bone regeneration by bone marrow stem cells/fibrin glue composition. Arch Oral Biol, 2012, 57(3): 238-244.
21.	朱元正, 易陽艷. 3-D 成球培養 MSCs 的研究進展及臨床應用前景. 中國修復重建外科雜志, 2017, 31(4): 497-503.

1. Chang Q, Lu F. A novel strategy for creating a large amount of engineered fat tissue with an axial vascular pedicle and a prefabricated scaffold. Med Hypotheses, 2012, 79(2): 267-270.
2. 陽水發, 易陽艷. 脂肪干細胞構建可注射型組織工程脂肪的研究進展. 中國修復重建外科雜志, 2015, 29(2): 245-249.
3. 陽水發, 易陽艷, 黃艷, 等. 人脂肪干細胞與不同濃度纖維蛋白膠的相容性研究. 重慶醫學, 2017, 46(2): 165-168.
4. Murphy KC, Fang SY, Leach JK. Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell Tissue Res, 2014, 357(1): 91-99.
5. 陽水發, 易陽艷, 黃艷, 等. 腺病毒介導 GFP 基因標記人脂肪來源干細胞可行性研究. 中國美容整形外科雜志, 2016, 27(8): 495-498.
6. Cecchi F, Rabe DC, Bottaro DP. Targeting the HGF/Met signaling pathway in cancer therapy. Expert Opin Ther Targets, 2012, 16(6): 553-572.
7. Nakamura T, Mizuno S. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci, 2010, 86(6): 588-610.
8. 焦志勇, 羅敏, 李盛, 等. 肝細胞生長因子對人羊水干細胞遷移和黏附能力的影響. 中華細胞與干細胞雜志, 2013, 3(3): 139-143.
9. Liu J, Pan G, Liang T, et al. HGF/c-Met signaling mediated mesenchymal stem cell-induced liver recovery in intestinal ischemia reperfusion model. Int J Med Sci, 2014, 11(6): 626-633.
10. Abd EN, Abdulrahman M, Helal M, et al. Regenerative capacity of allogenic gingival margin-derived stem cells with fibrin glue on albino rats’partially dissected submandibular salivary glands. Arch Oral Biol, 2017, 82: 302-309.
11. Kim YS, Sung CH, Chung SH, et al. Does an Injection of Adipose-Derived Mesenchymal Stem Cells Loaded in Fibrin Glue Influence Rotator Cuff Repair Outcomes? A Clinical and Magnetic Resonance Imaging Study. Am J Sports Med, 2017, 45(9): 2010-2018.
12. Hao C, Wang Y, Shao L, et al. Local Injection of Bone Mesenchymal Stem Cells and Fibrin Glue Promotes the Repair of Bone Atrophic Nonunion In Vivo. Adv Ther, 2016, 33(5): 824-833.
13. Liu Z, Zhu Y, Zhu H, et al. Enhancement of posterolateral lumbar spine fusion using recombinant human bone morphogenetic protein-2 and mesenchymal stem cells delivered in fibrin glue. J Biomater Appl, 2016, 31(4): 477-487.
14. Mehanna RA, Nabil I, Attia N, et al. The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats. Biomed Res Int, 2015, 2015: 846062.
15. Yang Y, Zhang W, Li Y, et al. Scalded skin of rat treated by using fibrin glue combined with allogeneic bone marrow mesenchymal stem cells. Ann Dermatol, 2014, 26(3): 289-295.
16. Wen Q, Zhou C, Luo W, et al. Pro-osteogenic effects of fibrin glue in treatment of avascular necrosis of the femoral head in vivo by hepatocyte growth factor-transgenic mesenchymal stem cells. J Transl Med, 2014, 12: 114.
17. Mcduffee LA, Esparza GB, Nino-Fong R, et al. Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold. Cell Tissue Res, 2014, 355(2): 327-335.
18. Wang K, Guan Y, Liu Y, et al. Fibrin glue with autogenic bone marrow mesenchymal stem cells for urethral injury repair in rabbit model. Tissue Eng Part A, 2012, 18(23-24): 2507-2517.
19. Ferris D, Frisbie D, Kisiday J, et al. In vivo healing of meniscal lacerations using bone marrow-derived mesenchymal stem cells and fibrin glue. Stem Cells Int, 2012, 2012: 691605.
20. Zhang L, Wang P, Mei S, et al. In vivo alveolar bone regeneration by bone marrow stem cells/fibrin glue composition. Arch Oral Biol, 2012, 57(3): 238-244.
21. 朱元正, 易陽艷. 3-D 成球培養 MSCs 的研究進展及臨床應用前景. 中國修復重建外科雜志, 2017, 31(4): 497-503.

Chinese Journal of Reparative and Reconstructive Surgery

Construction of injectable tissue engineered adipose tissue with fibrin glue scaffold and human adipose-derived stem cells transfected by lentivirus vector expressing hepatocyte growth factor

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