Synthesis and characterization of PLGA/Collagen ...

Proceedings of the 6th International Conference on Nanostructures (ICNS6) 7-10 March 2016, Kish Island, Iran

Synthesis and characterization of PLGA/Collagen electrospun nanofiber mat S. Nokhsteha, A. M. Molavia,b, A. Sadeghi*,a, M. Khorsand-Ghayenia a

Materials Research Group, Iranian Academic Centre for Education, Culture and Research (ACECR), Mashhad branch, Mashhad, Iran b

Materials Engineering Department, Tarbiat Modares university, Tehran, Iran *

[email protected]

Abstract: Using of skin scaffolds is as the most effective method in regenerating and replacing of serious skin injuries, therefore many studies were accomplished to explore and improve the properties and operations of these scaffolds. Nanofiber structure of these scaffolds are the most functional due to mimicking the morphology of extracellular matrix (ECM) in the body. In this study, electrospinning method was used to synthesize Collagen/PLGA (poly (lactide-co-glicolide) acid) nanofiber composite. The morphology of scaffolds was inspected by scanning electron microscope (SEM). The average diameter of nanofibers was 200 nm and pore diameters were in the range of 4-30 µm. The cytotoxicity and cell responses also were investigated for human dermal fibroblast (HDF) cell lines. The results are indicative of effective in vitro function of nanofibers and so the scaffold is a good candidate for skin regenerations. Keywords (11 Bold): Tissue engineering, skin scaffold, composite nanofibers. biocompatibility


Introduction Several studies have been investigated to develop and promote tissue engineering scaffolds using natural and synthetic polymers [1]. Artificial skin made of electrospun nanofibers have shown great advantages such as high surface area and porosity which can increased cell adhesion migration and proliferation significantly [2]. Unique biological properties of collagen such as biocompatibility, biodegradability and hydrophilicity made it an attractive material for clinical applications. Recent studies have indicated that collagen matrices of electrospun nanofibers are able to accelerate wound repair [3-6]. Mechanical properties of electrospun collagen nanofibers can be improved by blending with synthetic polymers. Jin et. al [7] synthesized nanofibers gelatin/PLLCL (60:40). In Vivo studies shown that an accelerating trend in wound repairing. Fibroblast cells adhesion on electrospun gelatin/PLA (70:30) also was evaluated by Hoveizi et. al [8]. After 5 days, SEM images showed that the good cell adhesion and cell spread completely on the surface. In this study, nanofiber scaffold of collagen type I/PLGA was produced by electrospinning and hexafluoroisopropyl alcohol, HFIP, was used as the common solvent. Mechanical and morphological properties of the scaffolds were evaluated and the results showed proper cell attachment on the electrospun nanofibers.

Materials and Methods PLGA (Poly (Lactide-Co-Glycolide 90000-1260000 KDa) and 1,1,1,3,3,3 propanol (HFIP) were purchased from and bovine type I collagen was prepared Institute.

Acid)) (75:25, -hexafluoro-2Sigma-Aldrich, by Iran Pasteur

Nanofibers preparation

Figure 1. A, B) SEM images of PLGA/collagen nanofibers with 20% (w/v) concentration, and C) pore size diagram of scaffold.

FTIR analysis In order to ensure the presence of collagen, FTIR (Shimadzu 8400S, Japan) of the samples was carried out by KBr disc method shown in Fig 2.

For production of nanocomposite fibers, PLGA:collagen solution (4:1) with concentration of 20% w/v was prepared by HFIP solvent and stirred by a magnetic stirrer overnight at room temperature. Electrospinning process was carried out with 1 ml h-1 flow rate under 29 kV. The distance between the needle and the rotating cylinder was 17 cm and the rotating speed was 500 rpm. After that, to remove remaining solvent the scaffolds were placed in a desiccator.

Scanning electron microscopy Scaffolds were sputter coated (SC7620 sputter coater) and surface morphology was evaluated using a scanning electron microscope (SEM, LEO-VP 1450). The fiber diameters were measured and reported by ImageJ analysis software (Fig 1).

Figure 2. FTIR spectroscopy for collagen, PLGA and composite samples.

Porosimetry


Measuring the pores size and their distribution in the samples was performed using the mercury method by PASCAL-140.

Where W0 and Wt were initial weight and final weight, respectively.

Fig 4. Weight loss of composite nanofibers. Figure 3. Fiber diameters for composite nanofibers scaffold.

Results and Discussion Cytotoxicity assay Biocompatibility of resulting mats was evaluated by observing the number of human dermal fibroblast (HDF) on the samples using MTT (3-[4, 5-dimethylthiazol-2-yl]2, 5 diphenyltetrazolium bromide) assay. Fibroblast cells with the number of 2×104 cells/well were seeded onto 12well plate containing fiber mats and DMEM (Dulbecco's Modified Eagle) Medium supplemented with 10% fetal bovine serum (FBS) and 100 U mL−1 penicillin/100 μg mL−1 streptomycin. Then MTT solution was added to the culture medium and incubated for 2 h at 37 0C. After that, dimethylsulfoxide (DMSO) was added and eventually the absorbance on 1, 7, and 14 days was measured at 570 nm by Elisa plate Reader.

SEM images showed uniform and bead-free nanofibers. The average fiber diameter was measured in the range of 200±60 nm. In addition, Figure 2 represents FTIR spectra of collagen, PLGA and composite nanofibers. Comparison of the characteristic peaks in collagen [10] indicates the presence of it in composite nanofibers [11]. According to the results of porosimetry (Figure 3), the sample pore diameter ranged from 4-30 µm. The maximum pore size in the sample is in the size of 16 μm. The pore size is within the range reported for good fibroblast cell adhesion [12]. Figure 5 represents the absorbance intensity or the primary number of cells was increased with time. As shown, absorbance intensity was significantly increased over time, indicating cell proliferation and cytocompatibility of the scaffolds. The degradation behaviour of the composite nanofiber scaffold is shown in Figure 5. As it can be seen, in the first week the weight loss of the samples due to the relatively high hydrophilic collagen is high. However, over time, the amount of lost weight reduced and in a long time is almost constant.

Conclusion

MTT assay of fibroblast cells for composite nanofibers.

Biodegradation assay To study the rate of biodegradation, Samples was cut into square pieces (with size 10 mm × 10 mm) and immersed in 1 M PBS (pH 7.4) at 37 °C for 7, 14 and 28 days. PBS solution was then removed and the samples were washed with distilled water and dried in a desiccator. The weight loss was calculated by the following formula [9]. w w t weight  loss  0 100 w0

Nanofiber scaffolds of collagen/PLGA with electrospinning process were developed and studied their properties for medicine applications. The produced scaffolds with having sufficient morphological properties (fiber diameter, 200±60 nm and pore size, 4-30 μm) and cellular interactions can be a good candidate for skin tissue engineering applications.

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