Integrating biomaterial functionalization and 3D printing to recreate the extracellular matrix environment

The extracellular matrix (ECM) is a complex environment involved in supporting cellular functions and organizing tissue architecture. Replicating this environment is essential in tissue engineering to develop in vitro functional constructs capable of mimicking the physiological behaviour of native human tissues. Functionalized biomaterials are commonly employed to reproduce the structural and biochemical complexity of the ECM. In this work, a variety of biomaterials were functionalized in different ways to create a library aimed at closely mimicking meniscal tissue for regenerative medicine applications. Both synthetic and natural polymers were used, and multiple functionalization strategies were explored to achieve diverse crosslinking approaches. Gelatin and hyaluronic acid were initially functionalized with phenol groups, and enzymatically crosslinked hydrogels were developed for extrusion-based 3D bioprinting. Gelatin was selected as a biomaterial because it is a derivative of collagen, the main structural component of the ECM, and it contains adhesion motifs for cells. Hyaluronic acid was chosen due to its abundance as a polysaccharide in the ECM and its excellent swelling and water-retention properties. Various formulations were tested by combining different component concentrations, and the most promising candidates were selected for further studies, which included the functionalization of gelatin with glycans, key signalling molecules capable of guiding cellular behaviour. Three different glycans were employed, and their biochemical and mechanical properties were characterized and modelled. The biological effects of these glycans were subsequently assessed, revealing differences not only in cell morphology but also in the expression of specific molecules. Next, 3D printing using stereolithography was explored through two different approaches: methacrylation, considered the gold standard, and UV-induced click-chemistry photocrosslinking. For this second strategy, gelatin and hyaluronic acid were functionalized with thiol groups, which are essential for the crosslinking reaction. Various formulations were then 3D printed to evaluate their performance compared to extrusion-based techniques. The mechanical and biological properties of the resulting constructs were also systematically characterized. Regarding methacrylation, a modified approach was employed given its widespread use in various applications. Polyethylene glycol acrylate alone does not support favourable cellular behaviours, such as attachment and spreading. To address this, mannose terminal groups were introduced to investigate whether they could enhance these behaviours. The printability of the modified hydrogels was also evaluated following the incorporation of the sugar moieties. Following this strategy, PCL was functionalized with three different molecules to enhance its biological performance and crosslinking capabilities. Since the aim was meniscal regeneration and given that the biochemical composition of the ECM is crucial for guiding cellular behaviour and ECM production, the human meniscal ECM was further analysed to identify the most relevant biochemical, morphological and mechanical motifs. Paediatric and adult tissues were compared to characterize the physiological composition and to assess pathological changes associated with different conditions, particularly by comparing osteoarthritic changes in normal versus valgus knee configurations. The biomechanics of the menisci were also assessed using finite element analysis on models derived from MRI segmentation, to evaluate stress distribution and displacements under physiological conditions. These data are essential for designing scaffolds with appropriate mechanical properties. Finally, 3D printing was used to fabricate anatomically shaped healing caps for gingival tissue following dental implant placement by employing a biocompatible, non-adhesive resin.