Hydrogels are an important class of biomaterials, widely used in tissue engineering. With these hydrogels, scientists aim to replace injured or removed functional tissue, because these materials show physicochemical similarity to extracellular matrix (ECM). Their permeability for nutrients, as well as their high water content are accounting for their application in soft tissue regeneration. Cells require amongst other factors, defined structural properties of hydrogels, to properly attach and proliferate within a matrix. Consequently, 3D fabrication of scaffolds can be realized by Additive Manufacturing Techniques, such as Two-Photon Polymerization (2PP). Macromolecules with natural origin, having photopolymerizable groups (precursors) can be fabricated via 2PP with high spatial resolution. The applied near-infrared laser offers high transparency towards biological tissues, being able to realize high penetration depth and little photo-damage of native matrices. Despite research has introduced a variety of different state of the art precursors [meth(acrylates)], these are potentially irritant and cytotoxic, when used as scaffolds to mimic ECM. Within this thesis, recently developed photopolymerizable precursors, based on hyaluronic acid (HA) were synthesized via an enzymatic transesterification reaction using divinyl adipate. Compared to their cytotoxic counterparts [meth(acrylates)], vinyl ester modified HA precursors (HAVE) show promising biocompatibility. The introduction of thiols into the precursor formulation enables effective crosslinking under high conversion using the concept of thiol ene chemistry. The slow homopolymerization rate of HAVE can thereby be overcome and multifunctional thiols can be used to study their effect on hydrogel matrices. The contribution of HAVE macromers on viscoelastic properties of hydrogels was investigated via photorheology. Based on the characteristics of the storage modulus, the effect of macromer size, degree of substitution, macromer content and type of thiol component on the crosslinking density, as well as on the reactivity of the formulation was investigated. Synthesized HAVE macromers, with different chain length and number of polymerizable groups on the HA backbone allow the fabrication of hydrogels with tailored mechanical properties. Additionally, a new approach was studied, to use HAVE in combination with collagen as interpenetrating network. Finally, cells were encapsulated via 2PP in the presence of HAVE macromers and their survival was studied.