*2.2. Hyaluronic Acid Blends*

More control over gel properties and better recapitulation of the extracellular matrix are possible by combining HA with other natural polymers that confer mechanical strength, cell binding motifs, and change the microstructure of the gel. Gelatin is an ideal copolymer for HA as it provides structural support and RGD-integrin binding sites that allow cell adhesion and proliferation, unlike HA alone [32]. Gelatin-HA constructs have been studied extensively for regeneration of articular cartilage [33–35], wound healing [36–38], and even vocal fold repair [39] due to their chondrogenic, angiogenic, and cell adhesive properties, and their tunable viscoelastic properties. Like HA, gelatin can be methacrylated, which allows for photopolymerization of gelatin-gelatin or gelatin-HA crosslinks. Constructs

made of methacrylated gelatin and HA have been shown to suppress hypertrophy and increase GAG expression by embedded, human bone marrow stem cells and, when tested in a full thickness osteochondral defect in rabbits, showed good cartilage regeneration [33]. Methacrylated HA and methacrylated gelatin can also be 3D bio-printed and polymerized with embedded cells without affecting their viability or chondrogenic properties, making them a good platform for custom, patient-specific cartilage implants [35]. Feng, et al. have also shown that a slightly different chemistry involving thiolated gelatin and HAvinylsulphone can form hybrid microgels, generated from crosslinked microbeads, similar to the HA-only microgels mentioned previously. Human bone marrow stem cells encapsulated in HA-gelatin microgels showed high viability and chondrogenic potential. When injected subcutaneously in mice, the cell-laden gels formed smooth, elastic, cartilage-like tissue, and reduced hypertrophy and vascularization over the course of 8 weeks [34].

This ability to drive cell behavior is also augmented by the slow degradation rate of gelatin. While HA alone promotes angiogenesis, decreasing gel degradation rate and providing cell binding sites, via complexation with gelatin accelerates healing and decreases counterproductive inflammatory cell migration at wound sites [36]. When carbohydrazide gelatin was combined with HA-monoaldehyde, they formed an injectable gel that showed no toxicity when tested with human umbilical cord endothelial cells in vitro [36]. Further, gels tested using an ex vivo rat aortic ring assay showed endothelial invasion and microvascular extension into the gel at the aortic ring-gel interface, supporting the hypothesis that HA, which can be angiogenic on its own at the correct concentrations [36,40], can be enhanced with a polymer that presents cell binding sites and slows gel degradation.

In wound healing, where angiogenesis is critical, Ebrahimi, et al. showed that electrospun gelatin-HA constructs could accelerate healing of thermal burns in mice [41]. In contrast to most other natural polymers, gelatin constructs can also be electrospun to generate nanofibrous gels instead of standard monolithic ones. Nanofibrous scaffolds structurally mimic the fibrillar structure of the extracellular matrix, allow for cell adhesion due to the high surface area to volume ratio, allow oxygen to permeate, and allow cell waste to escape, all while inhibiting pathogen infiltration [42], making them excellent candidates for wound healing applications. Similar electrospun constructs have been made using gelatin and HA combined with chitosan, which showed success in a mouse model of wound healing [43] and rabbit models of alkali induced corneal burns [44]. All of these constructs reduced inflammation and improved healing, demonstrating the potential improvement of gelatin-HA construct using a nanofibrous structure. However, nearly all of them, with the exception of acetic acid-based gel systems, use harsh solvents for electrospinning and crosslinking, making their use cumbersome and potentially hazardous. In situations where complex functionalization of the scaffold is required, an easily modifiable polymer such as poly(caprolactone) [45] can be used to electrospin HA instead of gelatin. Poly(caprolactone) (PCL) has been used extensively in biomaterials, especially for electrospinning, but it lacks the cell signaling characteristics and hydrophilicity of HA. PCL electrospun scaffolds doped with HA and epithelial growth factor have been shown to promote cell infiltration while also up-regulating collagen and TGF-ß1 expression in vitro. In vivo, the HA-PCL gels, when doped with endogenous growth factors, showed regeneration of a thicker epidermis layer and formation of an organized dermis layer as well in a rat model of full thickness skin wound healing [42]. Like the HA-Gelatin electrospun constructs, this system also employs harsh solvents, such as chloroform, leaving room for improvement in the electrospinning of nanofibrous HA scaffolds [42].

Less commonly, HA-gelatin solutions have been investigated for regeneration of muscle tissue and as a model system for lung tissue. Gelatin and HA can both be functionalized with tyramine to allow for gelation using horseradish peroxidase and H2O2 [32,46]. C2C12 murine myoblasts seeded on these tyramine crosslinked scaffolds were shown to retain myoblast differentiation and myotube formation, while HA-only and Gelatin-only gels did not. HA gels supported spherical cell morphology due to lack of cell binding sites in HA, and gelatin gels showed dedifferentiation, as the gel collapsed under cell traction

forces [32]. Kumar, et al. also showed that tyramine-functionalized HA and gelatin could be spin-coated into membranes and seeded with cells to generate an in vitro model of the alveolar basal epithelium for lung-based research. The films supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. When laden with growth factors, the membranes also induced some epithelial differentiation in MSCs [37]. Taken together, this research is suggestive of the vast potential of HA blended with gelatin and other bioactive species for tissue regeneration. It also highlights the array of crosslinking and manufacturing modalities that are under investigation to produce fully functional HA-based tissue engineered constructs.

While HA-gelatin blends are promising materials, many other HA blends have been investigated and have also shown promise. Tyramine functionalization of HA has been studied in combination with silk polymers for tissue engineering constructs. Raia, et al. have shown that HA-tyramine and silk fibroin-tyramine can be covalently crosslinked to form tunable hydrogels that begin to approach relevant mechanical properties and overcome some of the inherent weaknesses of HA [47]. In this study, silk fibers formed di-tyrosine bonds via horseradish peroxidase, resulting in highly elastic gels containing crystalline regions of silk. Tyramine-substituted HA, on the other hand, formed weak hydrogels that degraded rapidly. Use of a combination of both biopolymers overcame these weaknesses and resulted in tunable scaffolds. HA concentration in the matrix allowed adjustment of gelation time, degradation rate, and water retention. HA only hydrogels degraded within 6 days, while silk gels retained 70% of their mass on day 6. Hybrid gels allowed for tuning rate of degradation within this range of 1–6 days [47]. Silk-HA gels also achieved 100% strain before breaking, versus 30% in HA-only gels. Silk-only and HA-only gels exhibited storage moduli of 2.27 ± 0.09 KPa and 0.55 ± 0.03 KPa, respectively, while hybrid gels achieved moduli slightly beyond this range, peaking at 3.85 ± 0.08 KPa [47]. Silk gels alone were shown to allow adhesion and promote proliferation of human MSCs and this property was conferred to silk-HA hybrid gels. HA-only hydrogels inhibited MSC growth, showing an unadhered, spherical morphology after one week [47]. Combining silk and HA in this gel construct augments HA with the mechanical strength and degradation properties necessary to support cell growth with fine control over gel mechanical properties.

Similarly, tunable hybrid gels have been developed using HA and agarose. In contrast to HA, agarose has good gelatinizing properties, but exhibits slow degradation, limiting its use in some tissue engineering applications, which often target replacement of the engineered scaffold with host tissue [48]. Chu, et al. have shown that grafting of HA to agarose activated with epichlorohydrin resulted in a scaffold that presented the same cell regulation motifs as HA alone but also supported cell adhesion and proliferation. The gels were shown to stimulate TNF-α secretion in RAW 264 macrophages and upregulate Collagen I and III secretion by 3T3 fibroblasts. Further, when tested in a murine model of full thickness wound healing, agarose-HA gels showed rapid healing when compared to controls over the course of 21 days, showing that HA can facilitate wound healing past 1 week when combined with a slowly-degrading polymer [49].

While this list of HA-polymer blends is not exhaustive, it does demonstrate the enormous potential and versatilely of HA. HA interacts with cell receptors that regulate inflammation, cell differentiation, and cell motility, making it useful for a myriad of tissue engineering applications. However, it forms weak gels alone and does not adhere to tissues or support cell adhesion through integrin receptors thought to be required for tissue regeneration. These weaknesses can be overcome by functionalizing and crosslinking HA or combining it with another polymer, such as silk fibroin, gelatin, collagen, agarose, or polycaprolactone, which can provide strength and cell binding sites. In an appropriate scaffold, HA has been shown to induce chondrogenesis, osteogenesis, and wound healing by driving stem cell behavior. The ubiquitous nature of HA within the body and the ease with which it can be functionalized and combined with other polymers fully supports continued exploration of HA for successful development of tissue engineered products. A
