The usage of gelatin hydrogel is limited due to its instability and poor mechanical properties, especially under physiological conditions. Divalent metal ions present in gelatin such as Ca2+ and Fe2+ play important roles in the gelatin molecule interactions. The objective of this study was to determine the impact of divalent ion removal on the stability and mechanical properties of gelatin gels with and without chemical crosslinking. The gelatin solution was purified by Chelex resin to replace divalent metal ions with sodium ions. The gel was then chemically crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Results showed that the removal of divalent metal ions significantly impacted the formation of the gelatin network. The purified gelatin hydrogels had less interactions between gelatin molecules and form larger-pore network which enabled EDC to penetrate and crosslink the gel more efficiently. The crosslinked purified gels showed small swelling ratio, higher crosslinking density and dramatically increased storage and loss moduli. The removal of divalent ions is a simple yet effective method that can significantly improve the stability and strength of gelatin hydrogels. The in vitro cell culture demonstrated that the purified gelatin maintained its ability to support cell attachment and spreading.
Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nanofibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) were used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm–thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter was generated after removing the cellular components from the detached fibroblast sheet. The elastic modulus of the scaffold was well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) showed the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold’s biocompatibility was further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induced a significantly lower immune response compared to its unaligned counterpart, as detected by the pro-inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues.
Nitric oxide (NO) plays an important role in cardiovascular homeostasis, immune responses, and wound repair. The pro-angiogenic and antimicrobial properties of NO has stimulated the development of NO-releasing materials for wound dressings. Gelatin, an abundant natural biodegradable polymer derived from collagen, is able to promote wound repair. S-Nitroso-N-acetylpenicillamine (SNAP) can release NO under physiological conditions and when exposed to light. The objective of this project was to fabricate a NO-releasing gelatin-based nanofibrous matrix with precise light-controllable ability. Results showed that under controlled phase separation fabrication conditions, the gelatin formed a highly porous matrix with the nanofiber diameter ranging from 50 to 500 nm. Importantly, the removal of the trace amount of divalent metal ions within gelatin generated a more stable nanofibrous structure. N-acetyl-D-penicillamine (NAP) was functionalized onto the matrix and nitrosated with t-butyl nitrite, yielding a SNAP-gelatin matrix. Analysis of the photoinitiated NO-release showed that the SNAP-gelatin matrices released NO in a highly controllable manner. Application of increasing light intensities yielded increased NO flux from the matrices. In addition, the dried matrices stored in dark at 4 °C maintained stable NO storage capacity, and the purified (ion-removed) gelatin preserved higher NO-releasing capacity than nonpurified gelatin. The antibacterial effect from the SNAP-gelatin matrices was demonstrated by exposing Staphylococcus aureus ( S. aureus ) to a light-triggered NO flux. This controllable NO-releasing scaffold provides a potential antibacterial therapeutic approach to combat drug resistant bacteria.
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