PMC:4996398 / 17436-24839 JSONTXT

{"project":"2_test","denotations":[{"id":"27600217-19201462-69475904","span":{"begin":416,"end":418},"obj":"19201462"},{"id":"27600217-21193068-69475905","span":{"begin":504,"end":506},"obj":"21193068"},{"id":"27600217-11587588-69475906","span":{"begin":669,"end":671},"obj":"11587588"},{"id":"27600217-22336294-69475907","span":{"begin":672,"end":674},"obj":"22336294"},{"id":"27600217-22281421-69475908","span":{"begin":967,"end":969},"obj":"22281421"},{"id":"27600217-21334064-69475909","span":{"begin":1417,"end":1419},"obj":"21334064"},{"id":"27600217-18540674-69475910","span":{"begin":1420,"end":1422},"obj":"18540674"},{"id":"27600217-21112626-69475911","span":{"begin":1423,"end":1425},"obj":"21112626"},{"id":"27600217-9916770-69475912","span":{"begin":2044,"end":2046},"obj":"9916770"},{"id":"27600217-9916770-69475913","span":{"begin":2419,"end":2421},"obj":"9916770"},{"id":"27600217-12218178-69475914","span":{"begin":2422,"end":2424},"obj":"12218178"},{"id":"27600217-11857427-69475915","span":{"begin":3768,"end":3770},"obj":"11857427"},{"id":"27600217-11857427-69475916","span":{"begin":5111,"end":5113},"obj":"11857427"},{"id":"27600217-25067913-69475917","span":{"begin":5114,"end":5116},"obj":"25067913"},{"id":"27600217-25067913-69475918","span":{"begin":5249,"end":5251},"obj":"25067913"},{"id":"27600217-19126683-69475919","span":{"begin":5575,"end":5577},"obj":"19126683"},{"id":"27600217-15376189-69475920","span":{"begin":7398,"end":7400},"obj":"15376189"}],"text":"2.3. Alginate Derivatives\nAlginates can be chemically functionalized to alter physicochemical and biological characteristics and properties. One example is mentioned in Section 2.2.2 related to preparation of covalently gelled matrices. One approach for tuning degradation of alginate hydrogels includes the use of covalently cross-linked methacrylated alginates where the linkages are hydrolytically degradable [43,44], or inclusion of linkages that can be cleavable by matrix metalloproteinases (MMP) [45]. Another approach to enhance depolymerization is by partial periodate oxidation where some of the residues along the chain are made degradable by β-elimination [46,47,48]. Other tunable properties are solubility, hydrophobicity, bioactivity etc. Due to the free hydroxyl and carboxyl groups distributed along the backbone, alginate is a suitable candidate for chemical modification, and these are presented in reviews by Yang et al. [49] and Pawar and Edgar [50]. The most important modifications of alginate hydrogels for use in combination with cells are related to the ability to tailor and control the type and degree of cell interactions. This can be achieved by covalently conjugating alginate with heparin binding peptides (HBP) or peptide sequences found in ECM proteins. Cell matrix interactions can thereby be enabled via the non-integrin receptor syndecan for HBP or integrins for ECM peptides [51,52,53,54]. ECM peptide coupled alginates will be discussed in more detail below.\n\n2.3.1. Peptide-Coupled Alginates\nThe ability to modify the chemical and physical properties of alginate is a highly compelling incentive for using alginates in tissue engineering and regenerative medicine applications [55]. Cell attachment peptides, especially the sequence RGD (arginine-glycine-aspartic acid), have been shown to improve cellular adaptability to matrices, and such is also the case with alginate. Using aqueous carbodiimide chemistry, alginate can be modified by covalently grafting peptide sequences to the alginate molecule [56]. The interaction of cells with biomaterials is often mediated through cellular receptors that recognize adhesion molecules at material surfaces. One common example of such an adhesion ligand is the RGD peptide sequence, and it has been shown that RGD-coupled alginates (Figure 3) have the ability to initiate biological interactions between alginate hydrogels and cells [56,57].\nFigure 3 Chemical structure of RGD-alginate (arginine-glycine-aspartic acid conjugated to sodium alginate) (prepared using ISIS Draw). As cells do not have receptors that recognize alginate, proliferation and differentiation of some cells within an alginate hydrogel do require signaling molecules and matrix interaction. Studies using an alginate-based scaffold for 3D cell culture (NovaMatrix®-3D) have shown the importance of the presence of the RGD peptide sequence for cell proliferation. Table 1 lists various established cell lines grown in the NovaMatrix®-3D cell culture system and if RGD was required for cell proliferation. The commercial RGD-alginate NOVATACH MVG GRGDSP was used at a concentration of 120 µM RGD obtained by dilution with non-RGD alginate. (NOVATACH MVG GRGDSP contains 0.02–0.04 µmole RGD/mg alginate, and the exact value is reported on the certificate of analysis.). For example, the non‑tumorigenic cell lines C2C12 (mouse myoblast) and MDCK (canine kidney) did not proliferate in the alginate hydrogel during the first few weeks of culture. However, if RGD was available through incorporation of RGD-alginate, then cells displayed rapid proliferation. The data [58] on cell proliferation of C2C12 myoblast cells corroborated the importance of RGD peptide on cell proliferation found by Rowley and Mooney [59]. Moreover, after several weeks of culture, the C2C12 cells started to proliferate and fuse into multinucleated myofibrils as seen in Figure 4A. Figure 4 shows confocal images of the spatial organization of different cell types that were vital stained with Calcein AM (Molecular Probes) while inside the gel [60].\nFigure 4 Cellular organization within the alginate gel obtained by confocal microscopy of vital stained (Calcein AM) cells. Panels A–D and E–F show cells cultured without and with RGD-alginate, respectively. C2C12: murine myoblasts, HT-29: human colorectal adenocarcinoma cells, V79-379A: Chinese hamster lung fibroblasts, L1210: murine leukemia suspension cells, NIH:OVCAR-3: human ovarian adenocarcinoma cells, HCT 116: human colorectal carcinoma cells. Magnification ×100, scale bar 100 µm. Adapted from [60].\nmicroarrays-04-00133-t001_Table 1 Table 1 The importance of RGD (120 µM) as RGD-coupled alginate for cell proliferation in NovaMatrix®-3D (reproduced from www.novamatrix-3D.com) [58]. 1 Cell proliferation evaluated during three weeks of culture (NOVATACH is a tradename for RGD-alginate from FMC BioPolymer). 2 NOVATACH MVG GRGDSP influence on cell morphology. 3 Cell proliferation was accelerated in presence of NOVATACH MVG GRGDSP. Increased cell proliferation has been seen by increasing density of RGD [59,61]. Variations in nanoscale organization of RGD was shown to influence adhesion, proliferation and differentiation of preosteoblasts [61], which can be obtained by diluting RGD-alginates with different RGD densities along the chain with regular alginate. The peptide densities in alginate hydrogels can be optimized dependent on the cell culture system, but they are mainly comparable to the RGD density of commonly used biological matrices. Fischbach et al. [62] presented the number of RGD molecules in tumors associated ECM to be 8.6 × 1016 per mL matrix, which equals 143 µM. In the literature, the reported densities of peptides are presented in different ways such as µg peptide/mg polymer, µmole peptide/mg polymer, fmole/cm2 (2D), degree of substitution based on monomer or molecule (chain).\nThe presentation of the RGD peptide sequence has been shown to be an important cue regulating cellular behavior. This was demonstrated by, for example, Hsiong et al. [63] who compared the behavior of MC3T3-E1 preosteoblasts, human bone marrow stromal cells (hBMSCs) and D1 stem cells encapsulated in alginate hydrogels containing either linear G4RGDSP or cyclic G4CRGDSPC. The results showed that linear RGD densities promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts), but not for the hBMSCs and the stem cells. However, osteoprogenitor differentiation of all cells was seen in the gels presenting the higher-affinity cyclic form of the adhesion ligand. As presented in a review of Perlin et al. [64], the affinity and selectivity for different types of integrin receptors varies among cell types and is dependent on the flanking amino acids of RGD, the conformation and the length of the peptide sequence. Some integrins are cell type specific, whereas others mediate different cellular behaviors such as adhesion and migration. Hence, the type of optimal RGD containing peptide sequence and RGD density will vary depending on the study, which must be seen in combination with the physical properties of the peptide-containing alginate. Other extracellular matrix peptide sequences in addition to RGD have been found to be of interest to couple to alginate such as REDV (found in fibronectin), YIGSR (found in laminin) and VAPG (found in elastin) [51,65].\n"}