Bioinspired liver scaffold design criteria

Abstract

Maintaining hepatic functional characteristics in-vitro is considered one of the main challenges in engineering liver tissue. As hepatocytes cultured ex-vivo are deprived of their native extracellular matrix (ECM) milieu, developing scaffolds that mimic the biomechanical and physicochemical properties of the native ECM is thought to be a promising approach for successful tissue engineering and regenerative medicine applications. On the basis that the decellularized liver matrix represents the ideal design template for engineering bioinspired hepatic scaffolds, to derive quantitative descriptors of liver ECM architecture, we characterised decellularised liver matrices in terms of their biochemical, viscoelastic and structural features along with porosity, permeability and wettability. Together, these data provide a unique set of quantitative design criteria which can be used to generate guidelines for fabricating biomaterial scaffolds for liver tissue engineering. As proof-of-concept, we investigated hepatic cell response to substrate viscoelasticity. On collagen hydrogels mimicking decellularised liver mechanics, cells showed superior morphology, higher viability and albumin secretion than on stiffer and less viscous substrates. Although scaffold properties are generally inspired by those of native tissues, our results indicate significant differences between the mechano-structural characteristics of untreated and decellularised hepatic tissue. Therefore, we suggest that design rules - such as mechanical properties and swelling behaviour - for engineering biomimetic scaffolds be re-examined through further studies on substrates matching the features of decellularized liver matrices.

Keywords: ECM-mimicking scaffold; decellularisation; design criteria; hepatic cells; liver; tissue engineering.

Figure 1

Immuno-histochemical staining of the fresh (left column) and decellularized (right column) liver. Insets within each micrograph show a magnification to better display collagen, laminin and fibronectin localization. In both fresh and decellularised negative controls (CTR) where the primary antibodies were omitted, immunostaining was not detected. Collagen (COL) immunoreactivity was evenly distributed in the fresh liver parenchyma, laminin (LAM) was mainly localised in the extracellular spaces, while fibronectin (FIB) was found in the hepatocyte cytoplasm, membranes and extracellular spaces. The decellularized samples also showed positivity to collagen, laminin and fibronectin. Scale bars = 50 µm in the main micrographs and 10 µm in the insets.

Figure 2

Confocal acquisitions of an eosin-stained NI3 liver decellularised matrix. A) 3D rendering of a 62 x 62 x 16 μm volume and B) detail of a central slice of the confocal scan showing a rich intra-lobular network with an average (equilibrium swollen) pore size of about 22 μm.

Figure 3

Mass swelling behaviour of NI 3 decellularised liver matrices. Black circles represent mean swelling ratio values (Q) at different time points, while error bars denote respective standard deviation. * = significant differences between samples (one-way ANOVA, p < 0.05).

Figure 4

Examples of experimental LVR stress-strain data collected at various strain rates for NI3 liver dECMs. Decellularised liver LVR extended up to 3% strain and the apparent compressive modulus markedly increases with applied strain rate, as expected for viscoelastic materials.

Figure 5

HepG2 viability (a) and albumin secretion (b) measured at different time points (day 1, 3, 7) on collagen scaffolds with different viscoelastic properties (here coded according to their GTA crosslinker concentration, Table 2). Statistical differences are denoted with an asterisk (p < 0.05).

Figure 6

Immunofluorescent analyses of HepG2 cultured on collagen scaffolds with different viscoelastic properties (here coded according to their GTA crosslinker concentration, Table 2). Top row images show cell nuclei stained in blue (DAPI) at day 3. Cell clusters are highlighted with red circles. Bottom row images show HepG2 nuclei stained in blue (DAPI) and F-actin fibres stained in red (phalloidin) at day 7.

Figure 7

Experimental setup used for sorptivity measurements.