Electrospun SF/PLLA-CL graft with PRGF for inducing smooth muscle cell growth infiltration

摘要

Introduction: The construction of smooth muscle layer of blood vessel plays a key role in vascular tissue engineering. However, the penetration of smooth muscle cells (SMCs) into electrospun graft to form smooth muscle layer is usually quite limited due to small pore size and lack of inducing factor. Mere it is reported that the silk fibroin / poly (L-lactic acid-cc-E-caprolactone) (SF/PLLA-CL) vascular graft with big pore size loaded with platelet-rich growth factor (PRGF) can be fabricated by electrospinning. Previously, PRGF based graft were shown to have high cells growth rate. The purpose of this study was to investigate how deep the SMCs infiltration into graft by PRGF inducing. Materials & Methods: Materials used in this study consisted of SF, PLLA-CL, and PRGF. SF/ PLLA-CL (30/70, w/w) were dissolved in 1,1,1,3,3,3 hexafluoro-2-propanol (HFP; TCI America, Inc.) at a concentration of 120mg/mL to create the solutions used in the electrospinning process. PRGF was added in concentrations of 0 or 20mg of PRGF per milliliter of electrospinning solution, (SF/PLLA-CUPRGF(0), SF/PLLA-CL/PRGF(20)). To create vascular grafts, the prepared solution was loaded into a 5 mL plastic Becton Dickinson syringe with a 16 gauge blunt tip needle and dispensed at a rate of 3 mL/h. The needle tip was subjected to +30 kV with an air gap distance of 20 cm between the needle and the grounded mandrel. In order to study the infiltration and proliferation of cells on grafts, human aortic smooth muscle cells (SMCs, Cascade Biologics) were seeded on the grafts for 1,7 and 14 days. According to the previous methods[2], cryosections of SMCs-graft culture were stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) for imaging and examination of cellular infiltration. All samples were imaged using a Nikon TE300 microscope equipped with a 20× objective and a DXM 1200 digital camera was used to capture images. Ultraviolet light was used to capture DAPI stained nuclei. Cell depth was measured using Image-J software. Results and Discussion: SMCs were seeded in grafts for different days, and the results exhibited that the graft with PRGF enhanced SMCs growth and infiltration. After 14 days in vitro culture, SMCs still stayed on the surface of SF/PLLA-CL/PRGF(0) graft; in the mean while, SMCs kept growing steadily into the graft of SF/PLLA-CL/PRGF(20) from day 1 to day 14. SMCs penetrated into the graft, and the distance even reached 247 μm on day 14. It indicated that the PRGF could enhance SMCs growth as well as infiltration into graft. Table 1. Average depth results of SMCs penetration into grafts after 1,7 and 14 days of in vivo culture. Specimen Average depth of cellular penetration/(μm) Day1 Day7 Day14 SF/PLLA-CL/PRGF(0) ＜10 ＜15 ＜ 20 SF/PLLA-CUPRGF(20) 71±24 140±10 247±24 Fig. 1. Fluorescence microscopy images of cell infiltration over a period of 1,7 and 14 days. (Scale bar = 100 μm). Conclusions: The PRGF loaded graft can greatly promote SMCs' proliferation and induce cells' penetration, therefore it has potential to enhance smooth muscle layer construction ultimately.