Acceleration of tissue maturation by mechanotransduction-based bioprinting

The growing number of patients who require organ transplants, combined with the low number of organ donors, has resulted in organ shortages; therefore, the fabrication of human tissues and organs is an urgent need. However, the time required to fabricate an organ may result in risky delays for end-stage patients who urgently require transplants. During bioprinting, the maturation of the engineered tissue that is required before it is ready for implantation is lengthy. Here we use a previously introduced microscopic and mathematical model, the “zipper CAMs” (for ell dhesion olecules), to investigate the effective parameters involved in tissue dynamics. In our current study, we validated the ability of our model to accelerate the tissue maturation process. Our model shows that exploiting cellular mechanotransduction can accelerate post-printing tissue maturation. To verify this prediction experimentally, we devised a mechanotransduction-based bioprinter that accelerates the production of tissues by speeding up the fusion bioink particles. The mathematical microscopic model and the bioprinter described herein are expected to be highly useful in cell biology, tissue engineering, and biofabrication.

Figure 1

Schematic view of printing tubular structures such as blood vessels. (a) Tubular structures can be obtained by printing agarose (blue cylinders) and cylindrical cellular bioinks (red cylinders) layer by layer. (b) A closer look at the center of the printed constructs with cellular bioinks and agarose inside. (c) The continuous tissue construct forms through cellular self-assembly and the fusion of the discrete bioink particles. (d) By removing the agarose rod to which cells do not adhere, a tubular cellular structure is achieved. The inner diameter of the lumen can be controlled by the numbers and diameters of the printed cylindrical bioinks and agarose rods [14].

Figure 2

Bioprinting in the absence and presence of an external force: (a) Cellular bioinks were extruded according to the normal bioprinting protocol using flat surfaces to investigate the fusion without external forces. (b) Side view of two fusing cellular bioinks on a flat surface of agarose. (c) Six well dishes (3.5 cm diameter of each well) were used and covered by agarose to create flat surfaces. Cellular bioinks were printed in pairs in each well, and the fusion process was followed at different time points. (d)–(f) In mechanotransduction-based bioprinting, grooves are used to apply external forces on fusing cellular bioinks. Creating the glass foundation in 10-cm Petri dishes using capillary micropipettes (yellow cylinders). Glass tubes with 1.35-mm outer diameters were placed on the glass foundation, and the configuration was poured with liquid agarose. The grooves were created by removing the 1.35-mm outer diameter glass tubes and two cylinders were printed in each groove. (g) Bioprinting cellular bioinks inside grooves. (h) Side view of two fusing cellular bioinks. Printed cellular bioinks experience an external force. (i) Top view of printed cellular bioinks in the presence of grooves.

Figure 3

(a) Pairs of cylindrical bioinks of human skin fibroblast are printed in previously prepared grooves. (b) Schematics of two fusing cellular bioinks as well as a top view of a fusing pair cylindrical bioinks (at $\mathit{t}=0$) under a microscope. (c) Schematics of small pieces of fused bioinks as well as a top view of fused bioinks under a microscope. (d) Fused cylindrical bioinks are cut into small pieces using surgical scalpels and flipped ${90}^{\circ }$ in order to image the cross section of the fusion under a microscope attached to a camera. Essential parameters to investigate the fusion are introduced as $R, r$, and $\mathit{\theta }$. The fusion process will be studied quantitatively by plotting the $sin\mathit{\theta }$ vs time.

Figure 4

(a) Cross-sectional images of fusing cylinders at selected time points. The images are acquired by cutting small sections of fusing cylinders and flipping them by ${90}^{\circ }$ to image the cross section of the fusion area as depicted in Fig. 3. (b) $sin\mathit{\theta }$ vs time for cylindrical human skin fibroblast bioinks printed on flat agarose surface. Red circles represent the average of $sin\mathit{\theta }$ over six samples at each time point. The solid line is the theoretical fit of the data. The minimized residual sum of squares was 0.0098.

Figure 5

$sin\mathit{\theta }$ vs time for cylindrical human skin fibroblast bioinks printed onto grooves. Red circles represent the average of $sin\mathit{\theta }$ over five samples at each time point. The solid line is the theoretical fit curve. The minimized residual sum of squares was 0.0014.

Figure 6

The zipper model to investigate the rate of fusion. (a)–(c) The cascade of cellular attachments on the surface of two fusing cellular cylindrical bioinks can be considered as two series of closing zippers, one above and another one below the contact line. Analysis of the model in terms of statistical mechanics [32] provides the force and energy of fusion. (d) A series of zippers above the contact line. (e) Each zipper represents the attachment of consecutive cells on apposing cylindrical bioinks. The dashed curves represent the original locations of the adhesion molecules on the bioinks’ surfaces. The solid vertical line depicts the final closed zipper when the fusion of bioinks is complete. (f) Cell adhesion molecules on apposing bioinks can develop a bond or link. If the pair of adhesion molecules are far, the link is in configuration 1 with energy zero, whereas if they are sufficiently close [around $r_b$ in panel (h)], the link is in configuration 2 with energy $\varepsilon$. An external force may provide a biased cellular attachment using mechanotransduction for fusion. Red square: A closer look at the red circle in (f). Adhesion molecules can be considered springs with a deviation bond length; $|x_b-\lambda |$. (h) Molecules must be within an appropriate distance $\left(r_b\right)$ to be able to form a closed link.

Figure 7

The effect of deviation bond length on the forward rate constant. (a) 3D and contour plots for $exp\left[X\left(\alpha -\frac{1}{2}X\right)\right]$, when $0<\alpha ,X<1$. Here, we considered $|x_b-\lambda |=X$. As is shown, $exp\left[X\left(\alpha -\frac{1}{2}X\right)\right]$ can mathematically be greater than 1 for a range of $\alpha$ and $X$. For example, provided that the $\alpha$ is around 1, the greater values of $x$ also provide values greater than 1 for the exponential term. However, the real ratio for $\alpha$ to $x$ is 0.1 and 0.01, which are plotted in panels (b) and (c), respectively. It is clear that the smaller deviation bond length would produce greater values for the exponential term in Eq. (14)). This confirms the effect of external forces on accelerating the cell attachment process.

Figure 8

Schematic representation of some of the key mechanosensing molecules involved in the cell-extracellular matrix (ECM) interaction at the site of focal adhesion. Extracellular changes in stiffness, tension, or other mechanical stimuli are perceived by integrins, which undergo conformational changes and recruit focal adhesion kinase (FAK) to their vicinity on the intracellular side of the membrane. Other proteins aggregate with FAK to form the focal adhesion point (only a few of which are shown in this diagram); the aggregate signaling of these proteins influence the cell's actin cytoskeleton and vice versa.