*3.3. Processing Techniques and Manufacturing*

As tissue engineering systems become more advanced, research naturally trends towards improving their utility in the clinic. Recent studies using CS in tissue engineering, therefore, explore ways to make cell-seeded CS scaffolds injectable and tailorable to individual patients. Injectable tissue engineering constructs are advantageous as they do not require invasive surgeries to implant. Chen, et al., for example, have developed, an enzymatically crosslinked, injectable, and biodegradable hydrogel system comprised of carboxymethyl pullulan and chondroitin sulfate functionalized with tyramine. These conjugates are crosslinkable under physiological conditions using horseradish peroxidase (HRP) and hydrogen peroxide. Porcine articular chondrocytes embedded in these gels demonstrated proliferation and enhanced cartilage-like extracellular matrix deposition over controls, indicating chondrogenesis [79]. This HRP crosslinking method has the potential to form minimally invasive, injectable hydrogels for a myriad of tissue engineering applications, as the molecular weight ratios, polymer concentrations, and crosslinker concentration can all be modified to fine tune gel properties [80,81]. Li, et al. took this one step further and developed a similar system using oxidized CS and pullulan functionalized with adipic hydrazide that is self-gelling and forms in situ. Similarly, this system demonstrated good biocompatibility and chondrogenic properties [82], and supports the concept of developing in situ gelling CS scaffolds for tissue engineering.

A common way to make cell scaffolds injectable is to make them sheer thinning or thermo responsive via combination with a polymer like Chitosan. Chitosan is broadly used for the synthesis of injectable hydrogels due its biocompatibility [83] and thermosensitive capabilities [84]. CS has been combined with chitosan-poly(hydroxybutyrate-co-valerate) in the form of a nanoparticle for nucleus pulposus regeneration. Similar to CS + collagen systems, this hydrogel system supports viability, adhesion, and chondrogenic differentiation of adipose derived stem cells and shows potential for NP regeneration [85]. Alinejad, et al.'s work provides evidence that the gels made with chitosan and CS can be prepared with weak bases such as sodium hydrogen carbonate and beta-glycerophosphate to form thermosensitive, injectable and biocompatible scaffolds with tunable physical properties. Cytocompatibility of these hydrogels scaffolds was also shown to be good. When evaluated with L929 fibroblasts, they showed high viability and metabolic activity for up to 7 days. This effect was enhanced by the addition of CS relative to controls. [56]. CS can also be linked to a chitosan scaffold if the chitosan is functionalized with hydroxy butyl groups and the CS is oxidized, allowing them to crosslink via the Schiff-base reaction [58]. These injectable gels also show good biocompatibility and support adipose derived stem cells, while not eliciting an immune response [58]. This injectable system, however, differs, in that a pre-gel of oxidized CS and hydroxy butyl chitosan can be injected and subsequently completely gelled by injecting more oxidized CS. The authors see this as applicable in molding processes for custom made tissue engineering constructs that are shaped to the patient [58].

Injectable gels also open the door to 3D bioprinting of tailored, patient-specific constructs. Bioprinting generates 3D scaffolds with reproducible and complex structures and offers the opportunity to generate customized hydrogel scaffolds with a predetermined pattern, shape, and size. Engineered cartilage plugs, for example, can potentially be sized to a patient's joint and shaped exactly to match the defect they aim to repair. In order to 3D print a tissue engineering scaffold, the gel used must have the correct rheological properties to be extruded and undergo rapid gelation upon deposition [86]. Abbadessa, et al. have combined photopolymerizable methacrylated CS with thermosensitive poly(N-(2 hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10). Unlike polymer solutions composed of methacrylated CS alone or M15P10 alone, mixtures containing CS and M15P10 showed strain-softening, thermo-sensitive and shear-thinning properties. The 3D printing of this hydrogel resulted in the generation of constructs with tailorable porosity and embedded chondrogenic cells remained viable and proliferating over a culture period of 6 days [86] confirming the potential of this hydrogel solution for injectable, cell laden tissue engineering constructs.

While 3D printing and injection molding allow engineered tissues to be structurally modified on the macro scale, they do not offer the nanoscale structural control of electrospinning. As mentioned previously, electrospun, nanofibrous scaffolds have many advantages over monolithic hydrogels for some tissue engineering applications, namely for dermal grafts. They structurally mimic the extracellular matrix, allow for cell adhesion, allow oxygen to permeate, and allow cell waste to escape, making them ideal for wound healing [42]. Unmodified CS and the aforementioned hydrogel systems do not allow for electrospinning as do polycaprolactone-based systems [83]. Using acetic acid and water to reduce the use of potentially toxic organic solvents, Sadeghi, et al. have electrospun a gelatin/polyvinyl alcohol/chondroitin sulfate nanofibrous scaffold for skin tissue engineering [87,88]. Results indicated that the gels were not cytotoxic and L929 fibroblasts attach and proliferate on the scaffolds without issue, as assessed via scanning

electron microscopy [87], indicating they may be suitable for skin remodeling and regeneration. Further, this suggests that with further work, viable methods for electrospinning biocompatible scaffolds at scale will be realized.

In summary, Chondroitin sulfate-based gel systems have been developed for cartilage and bone repair, and wound healing due to their ability to direct cell attachment, cell lineage commitments, and differentiation [60,61]. Similar to HA, CS lacks the essential mechanical properties necessary for implantation. The bulk of current research, therefore, focuses on adding CS to bulk scaffolds for mechanical support or crosslinking CS. CS, when self-gelled or crosslinked to another organic or inorganic agent, has been shown to promote mineralization and osteogenesis, chondrogenesis, and wound healing in cell-laden tissue engineering constructs. Collectively, these studies demonstrate the key role CS plays in serving as a depot for growth factors to rapidly make them available as necessary for regeneration and engineering of new tissues. They also highlight the importance of highly charged sulfate groups on CS for binding of these factors and aggregation of ions such as the calcium and phosphate required for skeletal and dental bone mineralization. Many advances have also been made in making these constructs injectable and customizable using 3D printing and newer crosslinking modalities, while reducing the use of harsh, cytotoxic chemicals. A summary of CS hydrogel system and their tested behavior in vitro and in vivo can be found in Table 3.

**Table 3.** Summary of all aforementioned chondroitin sulfate hydrogel types and their tested behavior in vitro and in vivo.


