(368ae) Modifying a Thiolated Gelatin Hydrogel for Improved Mechanical and Biological Efficacy in a Multicompartment Tendon-to-Bone Scaffold for Regenerative Rotator Cuff Repair

Musculoskeletal conditions and injuries present a common, costly, and chronic challenge to the over 1 billion people affected worldwide. Such issues can severely limit quality of life, making musculoskeletal conditions a leading case of disability in the world. With over 4.5 million annual physician visits, shoulder pain represents the third most common type of musculoskeletal injury, and such pain is often associated with injury to the rotator cuff, a condition that remains a clinical challenge to repair. The rotator cuff connects soft tissue (muscle, tendon) to hard tissue (bone) through a fibrocartilaginous transitionary region known as the enthesis, providing critical mechanical support by dispersing stress and thus reducing potentially damaging stress concentrations. Unfortunately, following a tear, the highly structured and supportive fibrocartilaginous enthesis can be replaced with disorganized fibrovascular scar tissue. This tissue is more susceptible to inflammation and far weaker than the original tissue, heightening the risk of re-tear. While surgery can reattach torn tendon back to bone, it is not well-equipped to facilitate regeneration of the native enthesis, resulting in frequent surgical re-failure, reported as high as 94% in large and massive tears. Such challenges necessitate the need for next-generation techniques of rotator cuff repair, and we are developing a biomaterials-based approach to promote regeneration of the native enthesis. Our design integrates instructive tendon- and bone-mimetic scaffolds via a thiolated gelatin (Gel-SH) hydrogel, which mechanically supports the transition. This hydrogel also provides a region for biological transition, and we have recently shown that human mesenchymal stem cells (hMSCs) cultured within the osseous and tendinous regions of the material can significantly influence interfacial cellular behavior toward an entheseal phenotype through paracrine signaling. However, while the current material provides a permissive environment for enthesis engineering, it would benefit from greater enthesis-associated activity as well as an stronger mechanical properties (increased compression modulus, toughness). Here we outline our efforts to improve both the mechanical support offered by this hydrogel within the overall material and the biological cues it provides in guiding a fibrocartilaginous enthesis phenotype.

Methods

Multicompartment scaffolds are fabricated through the lyophilization of precursor solutions layered within a custom Teflon-copper mold. Precursors for the tendinous and osseous zones are created by homogenizing a slurry of collagen and chondroitin sulfate, as well as other salts and minerals, in an acidic buffer solution. Gelatin is thiolated by Traut’s reagent, dialyzed, lyophilized, and used to create a 3.5 wt% hydrogel precursor. Crosslinking is achieved enzymatically, using tyramine and horseradish peroxidase to oxidize thiol groups on the gelatin backbone, leading to the formation of disulfide bonds. The collagen slurries and hydrogel precursor are loaded into the mold, allowed time for hydrogel diffusion and crosslinking, and lyophilized; a copper wall facilitates unidirectional heat transfer on one side, creating aligned, elongated pores in the tendinous area of the scaffold while the osseous region retains an isotropic pore structure. Multicompartment material properties are evaluated via SEM imaging, pore size analysis, and uniaxial tensile testing. For in vitro work, scaffolds are sterilized via ethylene oxide treatment, hydrated in phosphate buffered saline, and exposed to additional crosslinking (EDC/NHS carbodiimide chemistry) before incubating in cell culture media. Human mesenchymal stem cells (P4-P5, RoosterBio) are cultured in flasks, seeded onto hydrated scaffolds, and evaluated over periods between 7 and 28 days for cell number (DNA isolation), metabolic activity (alamarBlue assay), gene expression (PCR or NanoString nCounter), and protein expression (Western Blot, ELISA, etc.). Such methods allow us to consider modifications to this system that improve overall scaffold stability and mechanics as well as bioactivity and relevance in enthesis tissue engineering.

Results

Biologically, we have demonstrated that this gelatin platform supports chondrogenic potential with the proper signaling (Fig. 1A), that hMSCs in neighboring osseous and tendinous compartments generate signals that significantly influence hMSC behavior within Gel-SH (Fig. 1B), and that this influence is also observed in the full multicompartment material. We are currently considering the incorporation of other naturally derived extracellular matrix components (chondroitin sulfate, collagen 2) to further enhance fibrochondrogenic capability of the material beyond the established baseline, and we are investigating the use of hMSC extracellular vesicles to strengthen tendinous and osseous tissue signaling. These modifications could allow for quicker and earlier signaling to occur, independent from the signals generated from seeded hMSCs. From a mechanical standpoint, we are exploring the use of ethylenediamine to induce greater amination of native gelatin, providing more reacting groups for the Traut’s reagent to thiolate and potentially resulting in greater material strength and toughness. Preliminary data (Fig. 1C) suggests that fabrication with this pre-aminated Gel-SH results in a stiffer overall multicompartment material. Through these modifications, we aim to develop a tougher material that provides faster and clearer signaling for hMSC differentiation, allowing for functional regeneration of the native enthesis for improved outcomes in rotator cuff repair.

Research Interests

Currently, my area of research is centered in the field of tissue engineering, combining biology and biochemistry concepts with approaches from chemical engineering and materials science. Prior to my graduate work, I conducted undergraduate research within the fields of biochemistry and biochemical development. These experiences have inspired my strong interest in work connected with the fields of biology and healthcare and nurtured my appreciation for process and product development. I love the notion of taking a promising concept (a target for tailoring biological activity, a method for improved hydrogel toughness, a fabrication technique for higher-throughput production of a complex scaffold) and building it from the ground up, exploring first its potential and then its scalability and value to the field.

Broadly, I am interested in applying my knowledge of chemistry, biology, biochemistry, and materials science toward the creation of new products and/or processes that can create positive change in the world. My background in tissue engineering research has given me valuable insights into considering how a biological product can be used and what design factors must be considered and tested. Even a particularly niche field as biomolecular engineering is still incredibly diverse, and I am excited to explore the options available to me and learn how I might put my skills to best use.