Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters

Abstract

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.

Figure 1

(A) Synthetic scheme of L1-peptide-conjugated fibrinogen. (B) Preparation of fibrin hydrogel.

Figure 2

Rheological Parameters of FH. (A) YIGSR measurements and (B) A99 measurements. Data represent the elasticity of unmodified FH (○), YIGSR-conjugated FH (△), A99-conjugated FH (□), SGIYR-conjugated FH (▲), and RAD-conjugated FH (■). Each data point represents the mean ± SD (n = 3, p < 0.05).

Figure 3

Par-C10 salivary cell cluster formation organization on peptide-conjugated FH. (A) Unmodified FH, (B) SGIYR-conjugated FH, (C) RAD-conjugated FH, (D) YIGSR-conjugated FH, (E) A99-conjugated FH, and (F) a combination of YIGSR (50%)- and A99 (50%)-conjugated FH. Par-C10 cells grown on YIGSR and/or A99 peptide-conjugated FH formed round organized structures (white arrows). Scale bars represent 200 µm.

Figure 4

(A) Total cell number and (B) Par-C10 salivary cell cluster formation were calculated. A combination of the peptides (YIGSR 50% with A99 50%) showed an increase in cell attachment and Par-C10 cell cluster formation as compared to the unmodified FH. Each data point represents the mean ± SD (n = 9, *p < 0.05, **p < 0.01).

Figure 5

Intracellular calcium concentration measurements. The cells were stimulated with 100 µM carbachol (Cch). Then, images were recorded and analyzed using Leica Application Suite X software. Par-C10 cells plated on unmodified FH (A), YIGSR-conjugated FH (B), A99-conjugated FH (C), a combination of YIGSR (50%)- and A99 (50%)-conjugated FH (D), SGIYR-conjugated FH (E) and RAD-conjugated FH (F). (G) Par-C10 cells cultured on YIGSR-modified FH displayed increased [Ca²⁺]_i. Data are expressed as means ± SD, where *p < 0.01 indicates a significant difference from control (unmodified FH).

Figure 6

Confocal microscopy images show ZO-1 (green) as stained by Alexa Fluor 488-conjugated goat antirabbit secondary antibody, F-actin as stained by Alexa Fluor 568-conjugated phalloidin (red), and nuclei (blue) as stained by TO-PRO-3 iodide. Right images are merged images. All images were obtained and analyzed using a Carl Zeiss 700 LSM confocal microscope. Par-C10 cells plated on unmodified FH (A), YIGSR-conjugated FH (B), A99-conjugated FH (C), and a combination of YIGSR (50%)- and A99 (50%)-conjugated FH (D). Scale bars represent 50 µm. White arrows indicate lumen formation.