A Single-Step Self-Assembly Approach for the Fabrication of Aligned and Multilayered Three-Dimensional Tissue Constructs Using Multidomain Peptide Hydrogel

Abstract

Hydrogels are homogenous materials that are limited in their ability to form oriented multilayered architecture in three-dimensional (3D) tissue constructs. Current techniques have led to advancements in this area. Such techniques often require extra devices and/or involve complex processes that are inaccessible to many laboratories. Here is described a one-step methodology that permits reliable alignment of cells into multiple layers using a self-assembling multidomain peptide (MDP) hydrogels. We characterized the structural features, viability, and molecular properties of dental pulp cells fabricated with MDP and demonstrated that manipulation of the layering of cells in the scaffolds was achieved by decreasing the weight by volume percentage (w/v%) of MDP contained within the scaffold. This approach allows cells to remodel their environment and enhanced various gene expression profiles, such as cell proliferation, angiogenesis, and extracellular matrix (ECM) remodeling-related genes. We further validated our approach for constructing various architectural configurations of tissues by fabricating cells into stratified multilayered and tubular structures. Our methodology provides a simple, rapid way to generate 3D tissue constructs with multilayered architectures. This method shows great potential to mimic in vivo microenvironments for cells and may be of benefit in modeling more complex tissues in the field of regenerative medicine.

Modeling Hypoxia Induced Factors to Treat Pulpal Inflammation and Drive Regeneration

“…3). Application of H 2 O 2 did not disrupt the formation of the encapsulating MDPs, because these constructs remained intact throughout all experiments and cell constructs formed as described previously 23 .…”

“…Cells mixed into MDP penetrate the MDP hydrogel, spreading into a multi-layered construct and taking on a fibroblast-like morphology, while retaining viability during 1-2 weeks in culture. This demonstrates the ability of MDPs to support SHED cell populations, the configuration of which can be tuned by altering the density of the MDP hydrogels 23 . plated into 6-well plates, and treated with 1 mg/ mL LPS (Sigma-Aldrich).…”

“…1). These cells were then encapsulated into 0.04% W/V low concentration MDP and grown into threedimensional cell constructs as described previously 23 . Three-dimensional cell constructs were then exposed to H 2 O 2 (30% of stock) placed in the culture medium to simulate hypoxia in vitro or treated with phosphatebuffered saline as negative controls.…”

“…After exposure to H 2 O 2, 0.04% W/V MDP constructs encapsulating SHED cells were cultured overnight and then lysed creating decellularized MDP matrices. Decellularized MDP matrices were then lyophilized as previously described 23 , generating powdered MDP, which contained SHED cell products from constructs placed under hypoxic stress via exposure to H 2 O 2 and negative controls not exposed to hypoxic stress. Thus lyophilized MDPs from both groups served as an acellular biomaterial, containing secretory products from SHED cells in either condition (Fig.…”

“…SYBR Green master mixes (Bio-Rad, Hercules, CA). PCR conditions were performed in 96-well plates as previously described 23 : first step, 95 C for 5 minutes; second step, 95 C for 30 seconds, 60 C for 30 seconds, and 72 C for 35 seconds for 35 cycles; and third step, 72 C for 5 minutes. For the final step, a melting curve was used to verify the specificity of amplicons.…”