In this study, we aimed at generating 3‐dimensional (3D) decellularized bovine spinal cord extracellular matrix‐based scaffolds (3D‐dCBS) for neural tissue engineering applications. Within this scope, bovine spinal cord tissue pieces were homogenized in 0.1 M NaOH and this viscous mixture was molded to attain 3D bioscaffolds. After resultant bioscaffolds were chemically crosslinked, the decellularization process was conducted with detergent, buffer, and enzyme solutions. Nuclear remnants in the native tissue and 3D‐dCBS were determined with DNA content analysis and agarose gel electrophoresis. Afterward, 3D‐dCBS were biochemically characterized in depth via glycosaminoglycan (GAG) content, hydroxyproline (HYP) assay, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Cellular survival of human adipose‐derived mesenchymal stem cells (hAMSCs) on the 3D‐dCBS for 3rd, 7th, and 10th days was assessed via MTT assay. Scaffold and cell/scaffold constructs were also evaluated with scanning electron microscopy and histochemical studies. DNA contents for native and 3D‐dCBS were respectively found to be 520.76 ± 18.11 and 28.80 ± 0.20 ng/mg dry weight (n = 3), indicating a successful decellularization process. GAG content, HYP assay, and SDS‐PAGE results proved that the extracellular matrix was substantially preserved during the decellularization process. In conclusion, it is believed that the novel decellularization method may allow fabricating 3D bioscaffolds with desired geometry from soft nervous system tissues.
Ö Z B u çalışmada, Melia azedarach L. yeşil meyve ve yapraklarının sulu özleri iki farklı özütleme yöntemi kullanılarak elde edildi. Yeşil meyve ve yaprakların özütleme verimleri sırasıyla, demleme yöntemi için %24.11 ve %37.98; çalkalama yöntemi için ise %17.76 ve %27.00 olarak bulundu. Demleme yöntemi ile ilgili toplam fenolik içerik, yeşil meyve özü için 173.67±10.84 mg galik asit eşdeğeri (GAE)/g kuru ağırlık ve yaprak özü için 312.33±9.81 mg GAE/g kuru ağırlık cinsinden tespit edildi. Diğer yandan demleme yönteminde, yeşil meyve özü için 172.51±13.23 mg Trolox/L ve yaprak özü için ise 569.16±10.41 mg Trolox/L cinsinden antioksidan aktivite belirlendi. Özütlerin kimyasal bileşimini tanımlamak için gaz kromatografisi-kütle spektrometresi (GC-MS) analizi yapıldı. Özütlerin insan adipoz kaynaklı mezenkimal kök hücreleri (iAMKH'leri) üzerindeki sitotoksisite seviyeleri ticari olarak mevcut olan XTT testi ile değerlendirildi. Sonuç olarak, yeşil meyve özütünün iAMKH'leri üzerine yaprak özütünden daha fazla sitotoksik aktiviteye sahip olduğu bulunmuştur Anahtar KelimelerMelia azedarach L., toplam fenolik bileşikler, sitotoksisite, insan adipoz kaynaklı mezenkimal kök hücreler. A B S T R A C TI n this study, aqueous extracts of Melia azedarach L. green fruit and leaves were obtained using two different extraction methods. The extraction yields of the green fruit and leaves were found as 24.11% and 37.98% for the infusion method; 17.76% and 27.00% for the rotating method, respectively. The total phenolic content, related to the infusion method, was ascertained for green fruit extract 173.67±10.84 mg Gallic Acid Equivalent (GAE)/g dry weight and leaf extract 312.33±9.81 mg GAE/g dry weight. In other respects, antioxidant activity related to the infusion method was determined for green fruit extract 172.51±13.23 mg Trolox/L and leaf extract 569.16±10.41 mg Trolox/L. Gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify the chemical composition of the extracts. The cytotoxicity levels of the extracts were assessed on human adipose-derived mesenchymal stem cells (hAMSCs) using commercially available XTT assay. Consequently, it has been found that the green fruit extract has more cytotoxic activity than the leaf extract on hAMSCs.
Extracellular matrix (ECM)-based tissue engineering scaffolds have an essential role in promoting tissue regeneration. Nerve tissue engineering aims at facilitating the repair of permanent damage to the peripheral and central nervous systems, which are difficult to heal. For this purpose, a variety of biomaterials are being developed consisting of numerous synthetic and/or natural polymers to provide axonal reinnervation and to direct the growth of axons. Here, we present a novel protocol that enables to fabricate a 3-dimensional (3D) decellularized scaffold derived from the bovine spinal cord (BSC) ECM (3D-dCBS) for neural tissue engineering applications. In this protocol, a viscous ECMderived gel from BSC is prepared, molded, and chemically crosslinked with EDC/NHS (3D-CBS) before decellularization process. Decellularization of 3D-CBS is performed with 1% SDS to attain 3D-dCBS.As compared with other available methods, our protocol is a novel decellularization method that preserves a more significant part of the ECM. We believe that the mentioned protocol has the potential to produce a bioengineered scaffold from spinal cord tissue with desired geometry for regenerative medicine applications related to neural tissue engineering.
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