Introduction: The natural extracellular matrix (ECM) has been an attractive model for the design and fabrication of bioactive scaffolds for tissue engineering. In this study, gelatin (major protein in ECM) and elastin (major protein in elastic tissue) were conjugated into polyethylene glycol (PEG) hydrogel in order to mimic multiple functions of the elastic natural ECM for repairing injured tissue.
Materials: Gelatin (type A, 175 bloom, from porcine skin) and Traut’s reagent were purchased from Sigma, while Elastin soluble was purchased from Elastin Product Company. PEG diacrylate (8k) was synthesized according to published paper[1]. Neonatal human dermal fibroblasts (NHDFs) and fibroblast basic medium-2 with supplements were obtained from Lonza Bioscience Company.
Methods: In this strategy, the thiolation of gelatin (0.60 g) was accomplished by reacting with Traut’s reagent (60mg); the thiolated gelatin was then conjugated to one end of PEG diacrylate (PEGDA, 4 molar folder excess to the sulfhydryl amount, shown in Figure 1) by Michael addition reaction at pH 8.0. This new conjugation method is capable of achieving constant and reproducible the gelatin-PEG precursor product, while elastin-PEG-acrylate was synthesized by the same method and reaction ratio. Finally, NHDFs (2×106/mL precursor solution) were encapsulated into gelatin and elastin hybrid PEG hydrogel by crosslinking the remaining double bonds of the gelatin-PEG-acrylate and elastin-PEG-acrylate under UV light. For 3-D cell encapsulation, three types of PEG hydrogels were prepared by combining elastin-PEG-acrylate into Gelatin-PEG-acrylate (48mg/mL) at concentration of 0 mg/mL (GP), 30mg/mL (GEP30), and 45mg/mL (GEP45).
Results: Nuclear Magnetic Resonance Spectroscopy (NMR) peaks of the gelatin-PEG acrylate and elastin-PEG acrylate showed the typical peaks of PEGDA (δ 3.64p.p.m.) and Traut’s reagent (δ 2.59p.p.m.), confirming that conjugation was successful. The mechanical properties of hydrogels were studied by shear storage modulus, while GEP45 has the highest storage modulus because of higher acrylate content. The DNA amount in three kinds of hydrogels increased after 10days, while GEP45 had the highest DNA amount. From the live/dead images of cell encapsulated hydrogels (Figure 2), NHDFs in GEP30 and GEP45 were growing faster than GP because elastin can promote fibroblast proliferation. Interestingly, NHDF were growing faster in GEP45 than in GEP30 before day 7. Type I collagen and elastin were found to be secreted and deposited more inside GEP30 and GEP45compared with GP according to Figure 3.
Discussion: DNA analysis and Live/dead staining proved that covalently incorporating gelatin and elastin into PEG hydrogels encapsulate the fibroblasts in situ with high cell viability. Moreover, solubilized elastin can markedly improve NHDF attachment and proliferation because NHDFs has several receptors which can interact with covalently conjugated solubilized elastin. Figure 2 indicates that NHDFs in gelatin and elastin hybrid hydrogel can actively remodel the matrix by secreting their own set of ECM proteins, which will be promising in repairing damaged tissue.
Conclusion: In conclusion, the in vitro 3-D cell culture results proved that the covalently crosslinked gelatin and elastin hybrid PEG hydrogels can provide better cell response compared to gelatin PEG hydrogel and be an effective and suitable scaffold for the regeneration of elastic tissue.
This research is supported by Nanyang Technological University Academic Research Fund Tier 1 and the Singapore National Research Foundation
References:
[1] Tissue engineering: Part A, 2008, 14, 3,349-360.