4.1.5. Hyaluronic Acid

Hyaluronic acid (HA) is a linear non-sulfated polysaccharide made up of repeated disaccharide molecules in alternating patterns (D-glucuronic acid and N-acetyl-D-glucosamine). This pattern is linked through interchanging β-1,4 and β-1,3 glycosidic bonds. HA is a fundamental component of ECM that regulates various cellular biological processes, such as migration, adhesion, proliferation, and differentiation, through binding with a specific receptor on the target cell [167,168]. Owing to its exceptional biocompatible, biodegradable and non-immunogenic properties, HA is clinically used for drug delivery and tissue regeneration [9,169,170]. As being a natural extracellular component, HA mimics the typical ECM and could initiate signaling pathways responsible for osteogenesis [171,172]. Moreover, the physicochemical and biological properties of HA could be altered by chemical modification [173].

Through adjusting the crosslinker (PEGTA) density, a series of hydrogels with different biochemical and biomechanical properties were developed by utilizing a thiolfunctionalized HA and a thiol-functionalized recombinant human gelatin. Human BMM-SCs were cultured on the hydrogels with different stiffness (storage modulus (G0 ) and corresponding PEGTA concentrations, namely 0.15 kPa (0.25%), 1.5 kPa (1.75%), and 4 kPa (2.5%), in adipogenic and osteogenic conditions. Adipogenic differentiation was confirmed by gene expression of lipoprotein lipase (LPL), as well as PPARγ2, with similar LPL expression levels demonstrated on the hydrogels with varying stiffness, whereas PPARγ2 expression was markedly enhanced upon increasing hydrogel stiffness. Cells exhibited spindle-shape morphology on the 0.15 kPa hydrogel, while displaying elongated and cuboidal appearance, similar to osteoblasts on greater stiffness hydrogels. Human MSCs cultured on the 1.5 kPa hydrogel significantly expressed osteopontin, while those cultured on the 4 kPa hydrogel revealed a significant upregulation in the expression of the late osteogenic gene (bone sialoprotein) [174].

In a novel HA hydrogel platform, ligation of the HAVDI adhesive peptide sequence from the extracellular N-cadherin domain 1 and the RGD adhesive motif from fibronectin led to Rac1-GTP-dependent reductions in the attachment of myosin IIA to the focal adhesions. This lack of myosin IIA incorporation into focal adhesions hindered the maturation of these adhesions with increasing substrate stiffness (E = 5, 10, and 15 kPa) and thereby decreased traction force generation on the underlying substrate. These alterations in the mechanical state of the MSCs further reduced mechanosensitive YAP/TAZ translocation to the nucleus, herewith attenuating the signaling pathways involved in mesenchymal development, including cell proliferation and osteogenic differentiation [175].

An in vitro culture system for osteochondral tissue engineering was developed, using HA gels with various stiffness (G0 ranging from 10 to 45 Pa) attained by mixing Glycosil®, a thiol-modified hyaluronan gel with the crosslinking agent PEG at ratios from (1:1 to 7:1). The co-differentiation media (a ratio of 50% chondrogenic:50% osteogenic) proved to be suitable for appropriate chondrogenic and osteogenic differentiation of human MSCs. On the stiffest matrix (HA:PEG construct at a 2:1 ratio), the three chondrogenic markers (aggrecan, collagen II, and sox 9) were expressed by the differentiated human MSCs cultured for 21 days [176].

Moreover, human BMMSCs were initially entrapped in a HA carrying sulfhydryl groups and a hydrophilic polymer bearing both acrylate and tetrazine groups with the shear elastic modulus (G0 ) =180 ± 42 Pa. The stiffness of the matrix was increased (G<sup>0</sup> = 520 ± 80 Pa) by adding HA conjugated with multiple copies of trans-cyclooctene (TCO) to the human MSCs-laden gel culture media. The 3D matrix tagged with a TCO-celladhesive motif promoted the cells to undergo remarkable actin polymerization, changing from a rounded phenotype to a spindle morphology with long processes. After an additional seven days of culture in the modified media, quantitative analysis showed that RGD tagging enhanced cellular expression of matrix metalloproteinase 1, whereas it decreased the expression of tenascin C and collagen I/III. RGD tagging, however, was not sufficient alone to induce chondrogenic, adipogenic, fibroblastic/myofibroblastic, or osteogenic differentiation [177].

Photo-crosslinked methacrylated HA hydrogels incorporating fragmented polycaprolactone (PCL) nanofibers with compression modulus 3122.5 ± 43.7 Pa promoted osteogenic differentiation of adipose-derived stem/progenitor cells incorporated into the composite

hydrogel. The biomarkers collage type 1, ALP, and Runx2 were significantly expressed in the hydrogels containing nanofibers. In addition, the results of alizarin red staining confirmed osteogenic differentiation [178].
