**8. Summary and Future Directions**

GAGs are used in tissue engineering constructs to recapitulate the ECM and, thereby, drive stem cell differentiation or retention of phenotype of implanted cells. This allows them to be used as implants for regeneration of damaged tissue. Recent research in this field focuses on tissue engineering constructs for wound healing in skin and cornea, restoring damaged cartilage, such as articular cartilage and the NP, restoring bone, and neuronal regeneration. Research in the last five years generally focuses on three key areas: (1) overcoming the physical limitations of GAGs alone, by developing scaffolds that mimic the rheological properties of native tissues that can be doped with CS to present its moieties; (2) exploration of growth factor and ion sequestering by GAGs in TECs and how this affects their ability to promote cell differentiation and tissue regeneration with one or multiple GAGs; (3) advancement of hydrogel crosslinking technologies to reduce cytotoxicity of components and reagents and confer new, useful properties, such as sheer thinning/thermos-responsiveness for 3D printing, or to allow for GAG-only scaffolds that do not require polymers such as chitosan, collagen, PEGDA, etc. Area (1) has been investigated quite heavily to date, but new potential applications are still emerging, and (2) and (3) leave much room for exploration. We have seen that incorporation of gel formations with one GAG to drive cell fate and tissue regeneration has been heavily explored in some tissues, such as cartilage and bone with some emerging research in combining multiple GAGs into a construct. Moreover, each GAG has been investigated to a different extent. The general trends in tissue engineering using GAGs and where each GAG stands in the process are summarized in Figure 3. Moving forward, we expect to see more tissue engineering constructs that incorporate multiple GAGs to elucidate their synergistic effects on stem cell fate and their composite potential for tissue regeneration, whether the form is as functionalized, crosslinked GAGs alone or GAGs immobilized or crosslinked into a gel composed of another polymer.

**Figure 3.** The four generalized steps of incorporating glycosaminoglycans into tissue engineering and the relative progress of each in this process.

All tissues contain a complex mix of different GAGs with different compositions, as described previously. It follows, then, that future research will increasingly focus on different "versions" of the same GAG and combinations of different GAGs in different ratios to recapitulate native tissue. This will be especially true as the wealth of knowledge regarding each GAG individually grows. Moving forward, we expect to see an increasing number of tissue engineering constructs with two or more GAGs and insight into their interplay. We also expect increased application of GAG based or GAG+ polymer gels in a wider variety of biological systems.

**Author Contributions:** All authors contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by: National Institutes of Health: T32 HL086350.

**Data Availability Statement:** No new data was created or analyzed in this study. Data sharing is not applicable in this article.

**Conflicts of Interest:** The authors declare no conflict of interest.
