Recapitulating the native tendon microenvironment through design of degradable, anisotropic engineered extracellular matrices

Project Summary
Following injury and repair, tendons rarely exhibit full restoration of function and reinjuries are prominent. Poor
clinical outcomes are due to deposition of disorganized scar tissue rather than tissue with the anisotropic,
hierarchical structure as in uninjured tendon. Prior work has demonstrated that anisotropic materials as
scaffolds to guide cells promote expression of tenogenic markers and provide a template for cell orientation,
showing promise for promoting regenerative healing. However, biomaterials used in these studies lack
tunability in biophysical and biochemical cues critical for fully recapitulating the native tendon environment in
an engineered extracellular matrix (eECM) and translation of the eECM to in vivo applications. To address
these limitations, we have developed a novel approach to fabricate anisotropic poly(ethylene glycol) (PEG)
hydrogel-based eECM to promote the alignment of human tenocytes. These anisotropic hydrogels are formed
using a two-stage polymerization that eliminates complications of using additives or complex processing
techniques to introduce anisotropy and enables scaling of the biomaterial without compromising material
properties. In the first stage, a network is formed via a Michael-addition reaction of 4-arm PEG-maleimide and
dicysteine peptides. The network is strained to introduce anisotropy, followed by a secondary thiol-ene
photocrosslinking of remaining peptide thiols and 8-arm PEG-norbornene to retain strain-induced alignment.
Notably, the eECM includes MMP-degradable crosslinks to balance structural cues and matrix remodeling. We
propose herein that hydrogel-mediated anisotropic guidance and biochemical cues to tenocytes in balance with
hydrogel remodeling will orchestrate native-like tendon deposition. In Aim 1, material properties of anisotropic
eECM will be characterized. Mechanical properties will be tested via tensile testing parallel and perpendicular
to alignment. Temporal retention of alignment as a function of MMP-mediated degradation will be analyzed via
wide angle x-ray diffraction. Human tenocytes will be seeded in the eECM and cell and matrix anisotropy will
be analyzed using microscopy. Matrix deposition will be analyzed by comparing ratios and organization of type
I to type III collagen deposition and gene expression of scleraxis, tenomodulin, mohawk, and ⍺SMA as
measures of regenerative versus fibrotic characteristics. Isotropic hydrogels will be used as controls with
analysis over 14 days in vitro. In Aim 2, tenocyte gene expression profiles induced by anisotropic, degradable
PEG hydrogel-based eECM will be comprehensively analyzed via RNAseq. Differential gene expression will be
assessed on day 14 samples to compare gene profiles between eECM groups and freshly isolated cells from
healthy and fibrotic tendons. Completing these aims will provide insight into materials design parameters for
eECM to develop a pro-regenerative environment for tenocytes. The hydrogels are expected to promote
alignment and suppress fibrosis, providing a platform for translating eECM as an effective 3D scaffold in
tendon repair and other aligned musculoskeletal tissues such as muscle.

Grant Number: 1R21AR084300-01A1
NIH Institute/Center: NIH
Principal Investigator: Danielle Benoit