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Bio cel ingl
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  3.1.4. Fibronectin Fibronectin is an important and ubiquitous ECM protein that resides in the SM. It mediates various effects (e.g. adhesion, migration, growth and differentiation) on many different cell types, in addition to being very important for vertebrate development, as evidenced by embryo-lethal knockouts [126]. Because of the numerous responses that it can elicit from a wide variety of cell types, many studies have chosen to isolate and use particular active domains that have been identi fi ed in the molecule. Of note is the RGD domain, which is involved in cell adhesion, migration and  proliferation [127]. Modi fi cation of 3D scaffolds with RGD sequences is able to improve the rate and degree at which MSCs migrate into and populate the scaffold [128]. Although it has been shown to not stimulate MSC proliferation [111], population of the scaffolds by MSCs may be explained by fi  bronectin ’ s ability to stimulate MSC adhesion and migration [99]. Similar RGD-modi fi ed scaffolds have also been shown to confer anti-apoptotic properties towards cells that migrate into the scaffold [129]. Apoptosis was apparent in non-modi fi ed scaffolds, suggesting that cell adhesion through the fi  bronectin RGD sequence is one method of cell preservation and survival. Exposure of MSCs to fi  bronectin results in increased activation of two therapeutic signaling pathways [130]: Akt, involved in survival [131,132], migration [133] and adhesion [134]; and, ERK, involved in proliferation [135], growth and differentiation [136,137]. The effect of fi  bronectin on these functions in MSCs may provide a molecular explanation for the results described in the aforementioned studies. The classical culture technique for producing EPC populations involves seeding whole isolates of peripheral blood mononuclear cells on fi  bronectin-coated plates and harvesting progenitorenriched  populations at the desired time point [33,35]. Fibronectin is thought to be adhesive for progenitor cells and a method for isolating EPCs through selective adhesion; however, if EPCs are cultured on fi  bronectin for an extended period, there is a dramatic switch to an endothelial phenotype, indicated by functional markers von Willebrand factor and vascular endothelial cell adhesion molecule [138], suggesting a potential role for fi  bonectin in EPC-to-endothelial differentiation. Immediately after exposure to fi  bronectin, MBs increase their transcription of muscle differentiation markers Pax7 and Myf5, but this increase is lost soon after the initial culture [103]. It is believed that fi  bronectin is adsorbed onto MBs and that this is a key method for controlling the switch from proliferative to differentiative  phenotypes [139]. Furthermore, the composition of the surrounding ECM affected the composition of the adsorbed fi  bronectin; MB- fi  bronectin cultures in the presence of collagen I displayed a greater increase in proliferation as compared to the same culture conditions on tissue culture polystyrene, attributed to substrate-induced conformational changes in fi  bronectin. Fibronectin can direct MB orientation [140], adhesion [141] andmyotube formation [142]. Although knock-down models still allow for myogenesis, the formed muscle exhibits disrupted fi  ber organization [143], suggesting the requirement for fi  bronectin in myogenic coordination. MBs may adhere to fi  bronectin throughits cellbinding sequence RGD [144], similar to other cell populations. During ischemia, skeletal muscle displays an increased potential for RGD sequence binding [145], suggesting a role for RGD-containing molecules in coordinating the regenerative response to ischemia. 3.1.5. Glycosaminoglycans Glycosaminoglycans (GAGs) are long unbranched polysaccharides that consist of repeating sugar dimers with diverse and numerous roles in connective tissues [146]. Three of the main divisions are: heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronic acid (HA). HA is the only GAG that is not bound to core  proteins. When a GAG is complexed with a core protein, such as glypican, perlecan or syndecan, it is referred to as a proteoglycan (PG). The types of GAG side-chain residues determine whether or not the molecule is a heparan or chondroitin sulfate proteoglycans. Much of the heterogeneity of proteoglycans is due to the wide range of sulfation patterns of GAGs. The side-chain residues are sulfated to different extents depending on the location, physiologic conditions, and source of production [147 e 149]. 3.1.5.1. Hyaluronic acid. Hyaluronic acid (HA) is a chief component  of the ECM in high-molecular weight chains. Shorter chains of the glycosaminoglycan HA have been shown to activate angiogenesis [150]. EPCs that have been cultured on HYAFF-11, a HA-based mesh, were protected from apoptotic cell death and also displayed increased proliferation, past 15 days in culture [151]. In addition, the HA-seeded EPCs were observed to adopt a spindle-shaped morphology, as well as to decrease their VEGF secretion, suggesting augmented EPC-to-endothelial differentiation. MSCs have also  been grown on HYAFF-11, and were observed to proliferate and to  produce and deposit ECM components, such as collagen I, collagen III, fi  bronectin, and laminin [152,153].  Native HA can have pro-survival effects on cells, such as by activation of the anti-apoptosis Akt pathway [154] or protection from toxic insult [155]. When HA-receptor interactions are inhibited, the HA-induced pro-survival effect is reversed [156], demonstrating a direct survival effect of HA. HA has also been used to successfully encapsulate EPCs, and when delivered to ischemic skeletal muscle, treatment with HA-EPCs had a more potent effect of restoring collateral circulation [155]. HA has been investigated as a co-polymer for silk fi  broin (SF)-  based scaffolds. The addition of HA to the SF-based materials supported enhanced proliferation of cultured MSCs [157]. The combined HA-SF scaffold also improved cellular in-growth to the material, and resulted in more ef  fi cient tissue formation, as measured by gene expression of cultured MSCs for collagens type I & III, and glycosaminoglycans. Together, these results suggest that HA may present itself as a suitable material in revascularization techniques, and may confer protective, anti-apoptotic effects, while supporting progenitor cell proliferation and differentiation. In vitro, expanded MSC populations have reduced abilities to adhere and migrate. It has been suggested that the reduced adhesion and migration of ex vivo-expanded MSCs could be reversed by supplementing cultures with HA [158]. Despite the improved functions that result from MSC-HA exposure, MSC incorporation into HA-based scaffolds is also known to induce chondrogenic differentiation and deposition of cartilage-like ECM [159,160]. Therefore, tissue engineers utilizing HA for skeletal myopathies must be particularly careful if selecting MSCs as the therapeutic cell source. To date, HA ’ s interactions with MBs have not been actively researched, perhaps because of its relatively low abundance in muscle ECM, compared to the ECM of HA-rich interstitia, such as cartilage. Of the few published studies, a clinically-relevant polyurethane for graft generation was modi fi ed with HA and seeded with MBs [161]. HA-incorporation had no effect on proliferation,  but it greatly increased the differentiation and maturation of seeded MBs. In a 3-dimensional HA culture, MBs displayed adhesion,  biocompatibility and growth [162]. 3.1.5.2. Heparan sulfate. Heparan sulfate proteoglycans (HSPGs) are  proteoglycans whose branches consist of heparan chains, or glucosamine and glucuronic or iduronic acid disaccharide units. HSPGs may be present on cell surfaces, and are reported to occur on endothelial cells [163 e 166]. A progenitor cell mobilizing and homing factor, stromal cell-derived factor-1 (SDF-1), is now known to be bound to cell surfaces by HSPGs [167,168]. Endothelial cells of different srcins can utilize different HSPGs; speci fi cally, bone marrow endothelial cells use a HSPG with a denser sulfation pattern than used by human umbilical vein endothelial cells, and are able to bind SDF-1 with a greater af  fi nity [169]. It is believed that this immobilization of SDF-1 can promote adhesion of EPCs, and that endothelial cells can exploit this in a method to direct  progenitor cells to a local site, such as ischemic muscle, where angiogenic cells are needed to aid in restoration of perfusion [170 e 172]. In addition to SDF-1, HSPGs are also known to bind other cytokines, such as fi  broblast growth factors, VEGF, and plateletderived growth factor [173], each of which is implicated in  progenitor cell migration, homing, and angiogenesis. It was recently described that HSPGs can support proliferation of MSCs [174]. HSPGs may have implicated roles in directing angiogenic stem cells for regeneration during ischemia, but they can also induce osteogenic differentiation of MSCs [175]. Another study has observed that HSPG can induce transdifferentiation of MBs into osteoblasts [176]. Therefore, as with HA, care must be taken to  avoid induction of osteogenesis, if one wishes to use HSPG for muscular tissue engineering. Despite this, it has been observed that HSPG content naturally increases during skeletal muscle regeneration [177], suggesting a to-be-de fi ned role for heparan sulfates in myogenesis. 3.1.5.3. Chondroitin sulfate. Chondroitin sulfate (CS) is a sulfated GAG consisting of alternating N-acetylgalactosamine and glucuronic acid monomers. It is widely recognized as an integral component of cartilage, whose mechanical strength is attributed to its CS content [178]. CS induces adhesive and proliferative phenotypes in MSCs [99], but also induces chondrogenic differentiation. CS is actively being researched for cartilage tissue engineering applications [179 e 182]. Despite this, our lab has demonstrated great success using a collagen I/CS blend for myopathic treatments consisting of: EPC delivery [16], restoring perfusion to ischemic skeletal muscle [19], and increasing the therapeutic effects of transplanted cells [92]. These studies did not observe evidence of chondrogenesis. This may be because MBs naturally produce CS [183] and this exposure therefore does not induce a chondrogenic response from muscles. Although the role of CS in muscle regeneration is not documented, CS may have a role in MB activity, as MBs produce CS [184] and its synthesis has been spatially and temporally correlated with the arrival of MBs during muscle development [185 
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