RGD-Modified Gellan Gum L. Stevensa,b, C. Ferrisa,b, E. Mumec, D. Kirchamajera,b, K. Gilmorea, I. Greguricc, S. Smithc, G. Wallacea and M. i. h. Panhuisa,b, aIntelligent Polymer Research InsDtute, ARC Centre of Excellence for Electromaterials Science, AIIM Facility, University of Wollongong, Wollongong, NSW 2522, Australia. bSoL Materials Group, School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia. cAustralian Nuclear Science and Technology OrganisaDon, Lucas Heights, NSW 2234, Australia.
Introduction Tissue engineering is a ﬁeld of research that has developed over several decades to now be on the cusp of providing treatments for a wide range of Dssue damage, from burns to organ failure. Materials used in Dssue engineering constructs are required to conform to very high standards of both performance and safety, requirements that are frequently applicaDon speciﬁc. As such there is an ongoing need for idenDfying new materials that combine strength, processability and favourable cell interacDons. Gellan gum is an anionic polysaccharide that forms ionic hydrogels under physiological condiDons. These materials are known to be cytocompaDble, but a lack of cell recogniDon sites limits its potenDal as a scaﬀold for aXachment dependent cells, which are a criDcal component of most Dssues. In this work we demonstrate a chemical modiﬁcaDon procedure for gellan gum, covalently linking the gellan gum chain to a pepDde sequence RGD, which has been shown mediate cell binding in similar systems1. The impact of modiﬁcaDon is examined using the aXachment dependent cell lines C2C12 and PC12.
Gellan gum hydrogels were formed by ionic cross-‐linking with Ca2+ added to DMEM culture media. These hydrogels were seeded with rat adrenal (PC12) and mouse skeletal muscle (C2C12) cell lines, which are widely used models of nerve and muscle cell behaviour, respecDvely. On unmodiﬁed gellan hydrogels (Fig 2: A,B,E,F) both cell lines were observed to form large cell clusters, with liXle observable interacDon with the hydrogel substrate. RGD-‐modifed surfaces (Fig 2: C,D,G,H) by contrast, enabled substanDally more cell spreading and encouraged typical proliferaDon cell morphologies, especially in the C2C12 cell line. Under diﬀerenDaDon condiDons (data not shown) C2C12 cells rapidly responded by contracDng into large ﬁbers, with some mulDnucleated myoﬁbers present by 5 days of diﬀerenDaDon. PC12 cells however, did not diﬀerenDate during exposure nerve growth factor, which typically prompts terminal diﬀerenDaDon. OpDmisaDon of the density of cell aXachment sites may be needed to facilitate PC12 diﬀerenDaDon.
Coupling Reaction Gellan gum was modiﬁed with the short pepDde sequence GGGGRGDSY by carboiimide chemistry similar to the procedure outlined by Rowley et al.2-‐3 for alginate modiﬁcaDon. The reacDve EDC intermediate is stabilised by the addiDon of a labile amine, SulfoNHS (Scheme 1), limiDng hydrolysis. The pepDde chain then binds through the terminal amine by subsDtuDon. It was found during iniDal reacDon aXempts that the divalent caDons in as received gellan inhibited EDC binding by occupying carboxylic residues on the gellan backbone. Gellan was thereaLer puriﬁed by heaDng gellan soluDons in the presence of Dowex 50W-‐X8 caDon exchange resin, followed by regeneraDon with NaOH. Ion content was assessed by ﬂame atomic absorpDon spectroscopy and found to have greatly reduced concentraDons of Ca2+ and Mg2+ (Table 1). When puriﬁed gellan gum was used, coupling eﬃciencies of approximately 40% were obtainable. The pepDde coupled gellan was reﬁned and isolated by dialysis against H2O, and lyophilisaDon.
Scheme 1 (Above): Simpliﬁed reacDon schemaDc for the coupling of the terminal amine of the GGGGRGDSY pepDde sequence with the carboxyl residues on the gellan gum polysaccharide.
Figure 2: Bright ﬁeld and calcien-‐stained ﬂuorescence microscopy images of PC12 (A-‐D) and C2C12 (E-‐H) cells aLer 24 hours of culture on hydrogels formed from puriﬁed (top row) and RGD-‐modiﬁed (lower row) gellan gum. The presence of the RGD sequence enhances cell-‐ surface interacDons and limits the formaDon of cell clusters. Under the test condiDons, this eﬀect appears to be more prominent in the C2C12 culture, which exhibited typical elongated phenotypes. PC12 cells did not diﬀerenDate, and retained rounded morphologies in the presence of nerve growth factor. Scale bars represent either 100 µm (D,F,H) or 200 µm (A,B,C,E,G)
Rheology and Printing One of the key beneﬁts of the gellan gum biopolymer, is it’s ability to rapidly form solid hydrogel structures through the addiDon of common divalent caDons such as Ca2+, enabling reacDve prinDng for scaﬀold formaDon (Fig 3). Rheological tesDng of gellan soluDons was conducted using an Anton Paar Physica MCR301 Figure 3: A freestanding structure printed using (w/v) puriﬁed gellan gum and small volumes Rheometer. Shear thinning behaviour was 1% of a concentrated soluDon CaCl observed for all tested samples, which is typical for polymer soluDons. Commercial gellan gum was substanDally more viscous than puriﬁed gellan, likely due to remnant divalent caDons forming ionic links between adjacent gellan polymer chains, limiDng ﬂuid ﬂow. Notably, the RGD modiﬁed gellan provided minimal resistance to ﬂuid ﬂow compared to puriﬁed gellan, possibly due to the pepDde chain 4: The shear dependent viscocity of 1% disrupDng gellan chains helix formaDon. Further Figure (w/v) soluDons of as received (blue), puriﬁed study of this eﬀect is needed to assess its (red) and RGD-‐modiﬁed (green) gellan gum. TesDng was conducted at 37˚C using a 50mm impact on RGD-‐gellan bioprinDng. small angle cone. 2.
Figure 1 (Right): The conjugaDon eﬃciencies for pepDde coupling reacDons under varied reactant condiDons. To measure reacDon products, the pepDde sequence was radiolabeled at the terminal tyrosine (Y) residue with a gamma emipng 125I using established methods4. RadioacDvity was detected with a Perkin Elmer Gamma counter, and converted to concentraDons using a calibraDon curve.
Table 1 (Below): ConcentraDons of four key anions in gellan gum soluDons determined by ﬂame absorpDon spectroscopy. As received gellan gum is primarily in the potassium form with a substanDal calcium component. Puriﬁed gellan gum is primarily in a sodium form, with no measurable calcium or magnesium.
Element (%w/w) Na+
Puriﬁed Gellan Gum
We have presented the synthesis and iniDal analysis of a new biomaterial for Dssue engineering, RGD-‐gellan gum. Through radiolabelling with I-‐125, the reacDon eﬃciency has been determined under a variety of condiDons. It was found that divalent caDons substanDally inhibit -‐COOH acDvaDon, and their removal by ion-‐exchange allowed for full reacDon eﬃciencies of around 40%. PC12 and C2C12 cell lines were both found to survive and proliferate on the RGD-‐modiﬁed material with less clustering than unmodiﬁed gellan surfaces, however C2C12 cells appeared to beneﬁt most from the RGD under the test condiDons, showing typical adherent and diﬀerenDated phenotypes. These results indicate that RGD-‐gellan has promise as a printable, soL cell scaﬀold for aXachment-‐dependent cells.
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