Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering

Abstract

Objective: As a biological tissue material, amniotic membrane (AM) has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional (3D) spongy scaffold made of the extracellular matrix (ECM) of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering.

Materials and methods: In this experimental study, cellular components of human amniotic membrane (HAM) were removed with 0.03% (w/v) sodium dodecyl sulphate (SDS). Quantitative analysis was performed to determine levels of Glycosaminoglycans (GAGs), collagen, and deoxyribonucleic acid (DNA). To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid (HCl 0.1 M) with pepsin (1 mg/ml). In the present experiment 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (H&E) staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scaffolds were cytocompatible.

Results: Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment (39 ± 4.06 μg/ml vs. 341 ± 29.60 μg/ml). Differences were observed between cellular and denude AM in matrix collagen (478 ± 18.06 μg/mg vs. 361 ± 27.47 μg/mg).With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% ± 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 μg/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 µm measured from scanning electron microscope (SEM) images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells.

Conclusion: The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation.

Keywords: Biodegradable; Extracellular Matrix; Skin Substitute.

Fig 1

Decellularization of human amniotic membrane (HAM): hematoxylin- and eosin (H&E)-stained native HAM (original magnification: ×20) Intact HAM (A), 0.03% (w/v) sodium dodecyl sulphate (SDS)-treated HAM (original magnification: ×20) (B), in each image, the arrows are indicating the apical surface of the HAM. Extracellular matrix (ECM) compositions were showed in intact AM, dendued AM and 3D AM scaffold (C, D) by using Russell-Movat staining (collagen, yellow) and (GAG, Green), Deoxyribonucleic acid (DNA) content of intact and denuded HAM was quantified using a micro plate fluorescence reader (E). Statistical differences between intact and denuded HAM groups; analysis of ECM components, including acid/ pepsin-soluble collagen, sulfated GAG (F, G). Statistical differences between collagen and GAG contents of intact HAM and 3D AM scaffold. (Data are shown as mean ± standard deviation), n=5 , A; P<0.001 and GAG; Glycosaminoglycan.

Fig 2

3D AM scaffold using Russell- Movat staining (collagen, yellow) and (GAG, Green) (A). Cross linked ECM derived AM scaffold produced by freeze dryer (B). SEM image of the surface (C). The cross section of the porous (D). PBS swelling ratio of ECM derived human AM scaffolds at different times (E). In vitro collagenase biodegradation; time course of weight remaining of ECM derived HAM scaffold, cross-linked with ratio (1:4) of NHS/EDC, after incubation in PBS containing 100 μg collagenase I, at 37˚C (F). Comparison results of effect of extract cytotoxicity of TCPs and scaffold groups on viability fetal fibroblast cells by MTS assay extract showed, (p>0.05) (G). (Data are shown as mean ± standard deviation). ECM; Extracellular matrix, AM; Amniotic membrane, GAG; Glycosaminoglycan, SEM; Scanning electronic microscopy, EDC; 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, NHS; N-hydroxysuccinimide, PBS; Phosphate-buffered saline, TCP; Tissue culture plates, n=5, A; P<0.001 and C; P<0.05.

Fig 3

SEM images of fetal fibroblast cells attached (arrows are indicating fibroblast cells) to ECM derived HAM scaffolds, after 7 days at surface (A) and internal surfaces of 3D spongy scaffold (B) obtained by cross sectioning. H&E images before and after seeding cells, The light microscopy images of H&E images showed the external surface of scaffold without cell (C) and attachment of human fetal fibroblast cells at external surfaces of scaffold, the arrows are indicating attachment of fetal fibroblast cells, the cells are dark grey and the AM scaffolds are light red (D). H&E images show the internal surface of the scaffold without cell (E) attachment and growth of fetal fibroblast cells at internal surface of scaffold after 7 days (F). MTS results showed the metabolic activities of fetal fibroblast cells in ECM derived HAM scaffold. Statistical differences in metabolic activity at days 7, 14 and 21 with 3D HAM scaffold in days 3 (G). SEM; Scanning electronic microscopy, ECM; Extracellular matrix, HAM; Human amniotic membrane, H&E; Hematoxylin and eosin. (Data are shown as mean ± standard deviation (SD). (n=5, A; P<0.001 and B; P<0. 01).