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.

The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized fibroblast cell sheets contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. They can serve as cell adhesion sites and mechanically strong supports for tissue-engineered constructs. An efficient method that can largely remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content during the decellularization process is critical. In this study, three decellularization methods were investigated: high concentration (0.5 wt%) of sodium dodecyl sulfate (SDS), low concentration (0.05 wt%) of SDS, and freeze-thaw cycling method. They were compared by characterization of ECM preservation, mechanical properties, in vitro immune response, and cell repopulation ability of the resulted ECM scaffolds. The results demonstrated that the high SDS treatment could efficiently remove around 90% of DNA from the cell sheet, but significantly compromised their ECM content and mechanical strength. The elastic and viscous modulus of the ECM decreased around 80% and 62%, respectively, after the high SDS treatment. The freeze-thaw cycling method maintained the ECM structure as well as the mechanical strength, but also preserved a large amount of cellular components in the ECM scaffold. Around 88% of DNA was left in the ECM after the freeze-thaw treatment. In vitro inflammatory tests suggested that the amount of DNA fragments in ECM scaffolds does not cause a significantly different immune response. All three ECM scaffolds showed comparable ability to support in vitro cell repopulation. The ECM scaffolds possess great potential to be selectively used in different tissue engineering applications according to the practical requirement.

There is a large demand for tissue engineered vascular grafts for the application of vascular reconstruction surgery or in vitro drug screening tissue model. The extracellular matrix (ECM) composition, along with the structural and mechanical anisotropy of native blood vessels are critical to their functional performance. The objective of this study was to develop a biomimetic vascular graft recapitulating the anisotropic features of native blood vessels by employing nanofibrous aligned fibroblast-derived ECM and human mesenchymal stem cells (hMSCs). The nanotopographic cues of aligned ECM directed the initial cell orientation The subsequent maturation under circumferential stress generated by a rotating wall vessel (RWV) bioreactor further promoted anisotropic structural and mechanical properties in the graft. The circumferential tensile strength was significantly higher than longitudinal strength in bioreactor samples. Expression of smooth muscle cell specific genes, α-smooth muscle actin and calponin, in hMSCs was greatly enhanced in bioreactor samples without any biochemical stimulation. In addition, employment of pre-made ECM and RWV bioreactor significantly reduced the graft fabrication time to 3 weeks. Mimicking the ECM composition, cell phenotype, structural and mechanical anisotropy, the vascular graft presented in our study is promising for vascular reconstruction surgery or in vitro tissue model applications.

Nitric oxide plays important roles in cardiovascular homeostasis, immune responses and wound repair. Therefore, polymers that release nitric oxide locally at the surface exhibit improved biocompatibility for biomedical implants through reducing neointimal hyperplasia and thrombosis caused by blood vessel wall damage. The objective of this article was to fabricate a nitric oxide–releasing gelatin hydrogel that can continuously generate nitric oxide at a physiologically relevant level and inhibit cell attachment and proliferation. The nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), was successfully conjugated to the gelatin hydrogel, which showed a rapid nitric oxide release in the first 2 h and then a slower but sustained release in the next 70-h period. Human mesenchymal stem cells (hMSCs), as a model cell line with wide biomedical applications, were used to examine the cell attachment and proliferation of the nitric oxide–releasing gelatin hydrogel. Compared with the control gelatin, the nitric oxide–releasing gelatin hydrogel demonstrated a 0·35 times lower hMSCs attachment at 6 h and a 3·15 times lower hMSCs proliferation after 72-h incubation. Moreover, hMSCs on nitric oxide–releasing gelatin exhibited a rounder cell shape and covered less cellular area than their counterparts on the control gelatin hydrogel. This gelatin hydrogel with local nitric oxide release at physiological level provides a promising therapeutic approach in enhancing the performance of biomedical implants.

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