Fabrication and Characterization of Spongy Denuded Amniotic Membrane Based Scaffold for Tissue Engineering

Objective: as a biological tissue material, amniotic membrane (am) has low immunogenicityand to date has been widely adopted in clinical practice. however, some featuressuch as low biomechanical consistency and rapid biodegradation is limited the applicationof am. therefore, in this study, we fabricated a novel three-dimensional (3d) spongy scaffoldmade of the extracellular matrix (ecm) of denuded am. due to their unique characteristicswhich are similar to the skin, these scaffolds can be considered as an alternativeoption in skin tissue engineering.materials and methods: in this experimental study, cellular components of human amnioticmembrane (ham) were removed with 0.03% (w/v) sodium dodecyl sulphate (sds). quantitativeanalysis was performed to determine levels of glycosaminoglycans (gags), collagen, anddeoxyribonucleic 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 wasused to improve the mechanical properties of 3d lyophilized am scaffold. the spongy 3d amscaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (h&e) staining,a swelling test, and mechanical strength and in vitro biodegradation tests. human fetalfibroblast culture systems were used to establish that the scafolds were cytocompatible.results: histological analysis of treated human am showed impressive removal of cellularcomponents. dna content was diminished after treatment (39 ± 4.06 ?g/ml vs. 341 ±29.60 ?g/ml). differences were observed between cellular and denude am in matrix collagen(478 ± 18.06 ?g/mg vs. 361 ± 27.47 ?g/mg).with the optimum concentration of 1 mmnhs/edc ratio1:4, chemical cross-linker agent could significantly increase the mechanicalproperty, and resistance to collagenase digestion. the results of 2, 4, 6-trinitrobenzenesulfonicacid (tnbs) test showed that cross-linking efficiency of am derived ecm scaffoldswas about 65% ± 10.53. scaffolds treated with nhs/edc cross-linker agent by 100?g/ml collagenase, lost 75% of their dry weight after 14 days. the average pore size of3d spongy scaffold was 160 ?m measured from scanning electron microscope (sem) imagesthat 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 proliferationin vitro. extracts and contact prepared from the 3d spongy scaffold of am showed asignificant increase in the attachment and proliferation of the human fetal fibroblasts cells.conclusion: the extra-cellular matrix of denuded am-based scaffold displays the mainproperties required for substitute skin including natural in vitro biodegradation, similarphysical and mechanical characterization, nontoxic biomaterial and no toxic effect on cellattachment and cell proliferation.