Three-dimensional printing of chemically crosslinked gelatin hydrogels for adipose tissue engineering

Abstract

Despite their outstanding potential and the success that has already been achieved with three-dimensional (3D) printed hydrogel scaffolds, there has been little investigation into their application in the regeneration of damaged or missing adipose tissue (AT). Due to their macroscopic shape, microarchitecture, extracellular matrix-mimicking structure, degradability and soft tissue biomimetic mechanical properties, 3D printed hydrogel scaffolds have great potential for use in aesthetic, structural and functional restoration of AT. Here, we propose a simple and cost-effective 3D printing strategy using gelatin-based ink to fabricate scaffolds suitable for AT engineering. The ink, successfully printed here for the first time, was prepared by mixing gelatin and methylenebisacrylamide (a crosslinker) to initiate the crosslinking reaction. The solution was then loaded into the cartridge (temperature T = 35 °C) of a pneumatic extrusion-based 3D printer and printed on a cooled surface (T = 4 °C) in the appropriate time window for ink printability as verified by rheological tests. Subsequently, the printed gelatin hydrogels were crosslinked at different temperatures to optimize their stability and fix the printed structure. The gelatin scaffolds crosslinked at 20 °C remained stable for 21 days at physiological temperature, with compressive mechanical properties mimicking those of AT (i.e. elastic modulus = 20 kPa). The 3D printed scaffolds showed no indirect cytotoxic effects on a 3T3-L1 pre-adipocyte cell line. Moreover, the printed scaffolds successfully promoted adhesion and proliferation of primary human pre-adipocytes, as demonstrated by LIVE/DEAD staining and Alamar Blue assay. The differentiation of primary human pre-adipocytes isolated from three different donors according to adipogenic phenotype was demonstrated by an increase in PPARγ gene expression detected by real-time PCR and accumulated lipid droplets stained by Oil Red O, thus proving the potential of the 3D printed gelatin hydrogels as scaffolds for AT engineering.

Plant Tissues as 3D Natural Scaffolds for Adipose, Bone and Tendon Tissue Regeneration

“…Control and decellularized apple samples ( n = 4, Ø = 10 mm, t = 2 mm) were soaked in PBS at 37°C for 1 week (i.e., Δw plateau) and then tested in the swollen state by applying one hysteresis compression cycle. Each test consisted in a load phase at a rate of 2.5% min –1 down to −30% strain, as previously reported for adipose tissue engineering, ( Chang et al, 2013 ; Contessi Negrini et al, 2020 ) and subsequent unload phase at a rate of 5% min –1 . From the stress-strain curves, the elastic modulus E (calculated as the slope in the 0–5% strain range), stiffness k (slope in the 15–20% strain range), maximum stress σ max , residual strain ε res and hysteresis area H (calculated as the area between the load and unload curves) were calculated.…”

Scaffold-based developmental tissue engineering strategies for ectodermal organ regeneration

“…In addition to incorporating fundamental features such as biocompatibility and biodegradability, classical TE scaffolds are designed to mimic the microarchitecture and mechanical properties of the mature tissue to be regenerated [ 9 , 10 ]. For instance, different scaffold designs include relatively stiff scaffolds with tuned porous structure for bone [ 11 , 12 ] and gradient porosity for osteochondral regeneration [ 13 , 14 ], scaffolds with preferential orientation for the regeneration of anisotropic tissues [ 15 , 16 ], or scaffolds with relatively soft mechanical properties and big pores for adipose tissue regeneration [ 17 , 18 ]. Classical TE strategies mainly aim at cell growth in a 3D structure and differentiation into the desired phenotype to form the mature tissue, pursued by guiding cell fate by modulating the scaffolds properties (e.g.…”

Modeling Adipogenesis: Current and Future Perspective

“…Although 3D models were used to study adipocyte functions, less is known about whether tissue microstructure and stiffness, as well as other ECM components, influence adipogenesis. Besides collagen, other materials and bioprinting technology might support the design and study of adipogenesis, for example using methylated or crosslinked gelatin [ 119 , 120 ]. However, they are widely used in translational medicine, rather than in the basic research.…”