*3.2. Chondroitin Sulfate Blends*

While methacrylated CS forms mechanically robust hydrogels that retain the inherent functionality of CS and have been shown to support chondrogenesis, osteogenesis and neurogenesis in vitro and in vivo, they require photopolymerization, meaning, in clinical

applications, these constructs would require specialized tools for delivery and ultra-violet light for polymerization, which could potentially injure surrounding healthy tissue [74]. Tang, et al. approached this issue by developing a hydrogel scaffold comprised of CS functionalized with graphene oxide. Johnson–Claisen rearrangement chemistry allowed graphene oxide (GO) to be functionalization with primary amines. A solution composed of CS and modified GO gelled in situ within 10 min and the incorporation of graphene improved stiffness and toughness drastically (320 and 70%, respectively) over gels made of just CS. They also proved to be highly porous, resistant to degradation, and enabled MSCs to proliferate and deposit collagen matrix [75]. Of note, however, the entrapment of cells and potential chemical remnants from the EDC/NHS chemistry did initially slow cell metabolism [75]. Potentially of value is the conductive nature of GO, which may confer the scaffolds with the ability to induce natural conductive currents to improve tissue regeneration.

New CS crosslinking methods provide new ways to study the interactions of CS with cells in the absence of other extracellular matrix components and, in that regard, are indispensable. However, CS is not the sole constituent of the extracellular matrix in any tissue in the human body, but rather is interlaced with other GAGs and natural polymers. Therefore, combining CS with different polymers presents an opportunity to further control a gel's rheological properties, present additional biological signals, and better mimic native tissue. For example, collagen scaffolds functionalized with CS have been shown to recapitulate the chondrogenic niche, modulate inflammation, and mimic the mechanical properties of native collagen [55]. Corradetti, et al. demonstrated that such constructs support chondrogenic differentiation in rat bone marrow-derived stem cells in vitro and suppressed inflammation in vivo. MSCs grown on CS-collagen constructs aligned with scaffold pores, whereas cells grown on scaffolds containing only collagen showed clustering behavior, demonstrating that the presence of CS in the CS-collagen scaffolds is essential to influence cell-scaffold adhesion and [55], therefore, cytoskeletal organization and differentiation [76,77]. These cells also developed more intracellular vesicles, which have been associated with enhanced intercellular communication [78]. The constructs innately induce chondrogenic differentiation, and even though they didn't support osteogenesis innately, they displayed a synergistic effect with osteogenic media, showing increased expression of osteogenic factors Alp, Spp1, and Bgla compared to controls [55].

CS has also been combined with collagen using genipin as a crosslinker for tissue engineered scaffolds for regeneration of different types of cartilage, such as the nucleus pulposus. Forming a lightly crosslinked, gelatin-like scaffold, type II collagen and CS crosslinked with genipin are biocompatible and support differentiation of adipose-derived stem cells in vitro. When used as an injectable carrier of adipose derived stem cells in a rat model of NP degeneration CS-collagen gels showed increased disc height, water content, proteoglycan and type II collagen synthesis, and partial recovery of NP structure [57].
