Tough and tunable scaffold-hydrogel composite biomaterial for soft-to-hard musculoskeletal tissue interfaces.

Tendon inserts into bone via a fibrocartilaginous interface (enthesis) that reduces mechanical strain and tissue failure. Despite this toughening mechanism, tears occur because of acute (overload) or degradative (aging) processes. Surgically fixating torn tendon into bone results in the formation of a scar tissue interface with inferior biomechanical properties. Progress toward enthesis regeneration requires biomaterial approaches to protect cells from high levels of interfacial strain. We report an innovative tissue reinforcement strategy: a stratified scaffold containing osseous and tendinous tissue compartments attached through a continuous polyethylene glycol (PEG) hydrogel interface. Tuning the gelation kinetics of the hydrogel modulates integration with the flanking compartments and yields biomechanical performance advantages. Notably, the hydrogel interface reduces formation of strain concentrations between tissue compartments in conventional stratified biomaterials that can have deleterious biological effects. This design of mechanically robust stratified composite biomaterials may be appropriate for a broad range of tendon and ligament-to-bone insertions.

Rheological Analysis of the Gelation Kinetics of an Enzyme Cross-linked PEG Hydrogel.

The diverse requirements of hydrogels for tissue engineering motivate the development of cross-linking reactions to fabricate hydrogel networks with specific features, particularly those amenable to the activity of biological materials (e.g., cells, proteins) that do not require exposure to UV light. We describe gelation kinetics for a library of thiolated poly(ethylene glycol) sulfhydryl hydrogels undergoing enzymatic cross-linking via horseradish peroxidase, a catalyst-driven reaction activated by hydrogen peroxide. We report the use of small-amplitude oscillatory shear (SAOS) to quantify gelation kinetics as a function of reaction conditions (hydrogen peroxide and polymer concentrations). We employ a novel approach to monitor the change of viscoelastic properties of hydrogels over the course of gelation (Δ tgel) via the time derivative of the storage modulus (d G'/d t). This approach, fundamentally distinct from traditional methods for defining a gel point, quantifies the time interval over which gelation events occur. We report that gelation depends on peroxide and polymer concentrations as well as system temperature, where the effects of hydrogen peroxide tend to saturate over a critical concentration. Further, this cross-linking reaction can be reversed using l-cysteine for rapid cell isolation, and the rate of hydrogel dissolution can be monitored using SAOS.

The influence of cyclic tensile strain on multi-compartment collagen-GAG scaffolds for tendon-bone junction repair.

Background: Orthopedic injuries often occur at the interface between soft tissues and bone. The tendon-bone junction (TBJ) is a classic example of such an interface. Current clinical strategies for TBJ injuries prioritize mechanical reattachment over regeneration of the native interface, resulting in poor outcomes. The need to promote regenerative healing of spatially-graded tissues inspires our effort to develop new tissue engineering technologies that replicate features of the spatially-graded extracellular matrix and strain profiles across the native TBJ. Materials and Methods: We recently described a biphasic collagen-glycosaminoglycan (CG) scaffold containing distinct compartment with divergent mineral content and structural alignment (isotropic vs. anisotropic) linked by a continuous interface zone to mimic structural and compositional features of the native TBJ. Results: Here, we report application of cyclic tensile strain (CTS) to the scaffold via a bioreactor leads to non-uniform strain profiles across the spatially-graded scaffold. Further, combinations of CTS and matrix structural features promote rapid, spatially-distinct differentiation profiles of human bone marrow-derived mesenchymal stem cells (MSCs) down multiple osteotendinous lineages. CTS preferentially upregulates MSC activity and tenogenic differentiation in the anisotropic region of the scaffold. This work demonstrates a tissue engineering approach that couples instructive biomaterials with cyclic tensile stimuli to promote regenerative healing of orthopedic interfaces.

High-level expression of basic fibroblast growth factor in transgenic soybean seeds and characterization of its biological activity.

The glycinin G1 gene encodes a soybean seed storage protein accumulating at a high level. We have used the G1 promoter to confer seed-specific expression of human basic fibroblast growth factor (bFGF) in transgenic soybeans. The coding region of 18 kDa bFGF was fused to the promoter or promoter-signal peptide sequence of G1 gene, and transferred into soybean. Analysis of transgenic plants demonstrated that bFGF transcript or protein was confined to the seeds. The highest level of bFGF accumulation in the seeds reached up to 2.3% of total soluble protein. The soybean-derived bFGF was biologically active as confirmed by its mitogenic activity on Balb/c 3T3 cells, and exhibited other properties identical to native bFGF. We also observed a seed-specific expression of beta-glucuronidase driven by the G1 promoter. These results indicated that the G1 promoter contains essential cis-elements for seed-specific expression, and thus can be used for expression of pharmaceutical proteins in soybean seeds.