Abstract
The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitute for soft tissue filling. In this study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin gene lentivirus. Our aim was to observe the effects of the insulin gene transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days culture of adipogenic induction. The accumulation of cytoplasmic droplets of neutral lipids was assessed by Oil Red O staining. The RNA and protein expression of transfected insulin gene in hUCMSCs were detected by QPCR and western blot. The effect of recombinant lentivirus transfection on the growth and proliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25 times higher than that in the untransfected group. The western blot showed that a positive band was discerned at the site of a relative molecular mass of 8 × 103 Dalton in transfected group. After adipogenic culture for 7 days, under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared in silk fibroin scaffold; round adipose droplets showed intracellularly; the size of the adipocytes was not homogenous, and the density of adipocytes in transfected group was significantly higher than that in untransfected group (P = 0.007, P 0.05). And there was also no significant difference in optical density (A) between cell group and cell-scalffold group (P = 0.066, P > 0.05). We concluded that insulin gene could obviously promote the adipogenic differentiation of hUCMSCs, and a tissue engineering adipose could be constructed by the silk fibroin scaffolds seeded with human insulin gene-modified hUCMSCs effectively in vitro.
Highlights
Soft tissue defect presents an ongoing challenge in plastic and reconstructive surgery after deep burns, trauma or tumor resection
RT-PCR results showed that the expression of adipogenic gene PPARγ-2 in transfected group was much stronger than that in untransfected group
We concluded that insulin gene could obviously promote the adipogenic differentiation of Human umbilical cord mesenchymal stem cells (hUCMSCs), and a tissue engineering adipose could be constructed by the silk fibroin scaffolds seeded with human insulin gene-modified hUCMSCs effectively in vitro
Summary
Soft tissue defect presents an ongoing challenge in plastic and reconstructive surgery after deep burns, trauma or tumor resection. Standard approaches to soft tissue reconstruction include autologous tissue flaps, autologous fat transplantation and alloplastic implants. All these approaches have disadvantages, including donor-site morbidity, implant migration and foreign body reaction. The limitations of autologous adipose transplantation for soft tissue defect have paved the way for the researchers to view mesenchymal stem cells (MSCs), with their inherent property of selfrenewal and multi-differentiation potential, as an alternate option [1] [2]. The key factors to construct tissue engineering adipose are seed cells and the cytokines which regulate the growth and differentiation of the stem cells. To promote the proliferation and differentiation of the stem cells (hUCMSCs), a plenti expression vector of pLenti6.3insulin-IRES-EGFP was established and was transfected into hUCMSCs in our laboratory; a foundation for the construction of tissue engineering adipose was settled [7] [8]
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