Julianne L. Holloway

A novel method for the direct fabrication of growth factor-loaded microspheres within porous nondegradable hydrogels: Controlled release for cartilage tissue engineering

Because of similar mechanical properties to native cartilage, synthetic hydrogels based on poly(vinyl alcohol) (PVA) have been proposed for replacement of damaged articular cartilage, but they suffer from a complete lack of integration with surrounding tissue. In this study, insulin-like growth factor-1 (IGF-1), an important growth factor in cartilage regeneration, was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles embedded in the PVA hydrogels in a single step based on a double emulsion. The release of IGF-1 from these hydrogels was sustained over 6 weeks in vitro. Poly(glycolic acid) (PGA) fiber scaffolds were wrapped around the hydrogels, seeded with chondrocytes, and implanted subcutaneously in athymic mice. The release of IGF-1 enhanced cartilage formation in the layers surrounding the hydrogels, in terms of the content of extracellular matrix components and mechanical properties, and increased integration between the cartilage layers and the hydrogels, according to gross observation of the cross-sections and histology. The compressive modulus of the cartilage-hydrogel constructs without IGF-1 was 0.07±0.02MPa, compared to 0.17-0.2MPa for hydrogels that contained IGF-1. The biochemical and mechanical markers of cartilage formation were not different between the low and high concentrations of IGF-1, despite an order of magnitude difference in concentration. This study shows that the sustained release of IGF-1 can enhance tissue formation and points to a possible strategy for effecting integration with surrounding tissue.

Mechanical evaluation of poly(vinyl alcohol)-based fibrous composites as biomaterials for meniscal tissue replacement

In this study, poly(vinyl alcohol) (PVA) hydrogels were reinforced with ultrahigh molecular weight polyethylene (UHMWPE) and PP fibers and evaluated as potential nondegradable meniscal replacements. An investigation of hydrogel and composite mechanical properties indicates that fiber-reinforced PVA hydrogels could replicate the unique anisotropic modulus distribution present in the native meniscus; the most commonly damaged orthopedic tissue. More specifically, fibrous reinforcement successfully increased the tensile modulus of the biomaterial from 0.23±0.02MPa without any reinforcement to 258.1±40.1MPa at 29vol.% UHMWPE. Additionally, the molecular weight between cross-links, bound water and the microstructure of the PVA hydrogels were evaluated as a function of freeze-thaw cycles and polymer concentration to lend insight into the processes occurring during synthesis. These results suggest the presence of multiple mechanisms as causes for increasing hydrogel modulus with freeze-thaw cycling, including hydrogen bonding between amorphous and/or crystalline regions, and the formation of highly concentrated regions of mostly amorphous PVA chains. It is possible that the formation of regions with highly concentrated amounts of PVA increases the load-bearing ability of the hydrogels.

The role of crystallization and phase separation in the formation of physically cross-linked PVA hydrogels

The biocompatibility, processing ease, and mechanical properties of freeze-thawed poly(vinyl alcohol) (PVA)-based hydrogels have encouraged significant research toward developing this material for various biomedical applications. Crystallization that occurs during the freeze-thawing process is cited in the literature as the primary mechanism responsible for the resultant mechanical properties. Further analysis, however, shows the presence of two unique mechanisms that contribute to PVAs mechanical properties. During freeze–thaw cycling water freezes causing phase separation, which facilitates crystallization. The impact of phase separation during freeze–thaw cycling was investigated by comparing freeze-thawed and aged PVA hydrogels. Aged hydrogels were not prepared by freezing and, therefore, did not exhibit significant phase separation. The amount of phase separation was discerned using optical microscopy in the hydrated state. Crystallinity and mechanical properties were also evaluated as a function of the number of cycles (for freeze-thawed gels) and aging time (for aged gels). For freeze-thawed hydrogels, crystallinity deviated significantly from the trend observed in compressive modulus, indicating that crystallinity was not the only factor determining the hydrogels mechanical properties. Phase separation was found to occur during freeze–thaw cycling independently of crystallization, especially at later freeze–thaw cycles (after the third). The trends observed for both crystallinity and modulus for aged hydrogels, however, were in better agreement with each other. Further evaluation of the mechanical properties of aged and freeze-thawed hydrogels with similar crystallinities indicated that freeze-thawed hydrogels have significantly higher modulus values (p < 0.05). As a result, phase separation, independently of crystallization, was determined to have a significant effect on gelation during freeze–thaw cycling. In particular, PVA-rich regions that are formed during phase separation, without additional cross-linking, are believed to have a significant effect on the resultant mechanical properties.

Modulating hydrogel crosslink density and degradation to control bone morphogenetic protein delivery and in vivo bone formation

Bone morphogenetic proteins (BMPs) show promise in therapies for improving bone formation after injury; however, the high supraphysiological concentrations required for desired osteoinductive effects, off-target concerns, costs, and patient variability have limited the use of BMP-based therapeutics. To better understand the role of biomaterial design in BMP delivery, a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel was used for BMP-2 delivery to evaluate the influence of hydrogel degradation rate on bone repair in vivo. Specifically, maleimide-modified HA (MaHA) macromers were crosslinked with difunctional MMP-sensitive peptides to permit protease-mediated hydrogel degradation and growth factor release. The compressive, rheological, and degradation properties of MaHA hydrogels were characterized as a function of crosslink density, which was varied through either MaHA concentration (1-5wt.%) or maleimide functionalization (10-40%f). Generally, the compressive moduli increased, the time to gelation decreased, and the degradation rate decreased with increasing crosslink density. Furthermore, BMP-2 release increased with either a decrease in the initial crosslink density or an increase in collagenase concentration (non-specific MMP degradation). Lastly, two hydrogel formulations with distinct BMP-2 release profiles were evaluated in a critical-sized calvarial defect model in rats. After six weeks, minimal evidence of bone repair was observed within defects left empty or filled with hydrogels alone. For hydrogels that contained BMP-2, similar volumes of new bone tissue were formed; however, the faster degrading hydrogel exhibited improved cellular invasion, bone volume to total volume ratio, and overall defect filling. These results illustrate the importance of coordinating hydrogel degradation with the rate of new tissue formation.

Design of semi-degradable hydrogels based on poly(vinyl alcohol) and poly(lactic-co-glycolic acid) for cartilage tissue engineering.

Articular cartilage damage is a persistent challenge in biomaterials and tissue engineering. Poly(vinyl alcohol) (PVA) hydrogels have shown promise as implants, but their lack of integration with surrounding cartilage prevents their utility. We sought to combine the advantages of PVA hydrogels with poly(lactic‐co‐glycolic acid) (PLGA) scaffolds, which have been successful in facilitating the integration of neocartilage with surrounding tissue. Through a novel double‐emulsion technique, PLGA microparticles and a high level of porosity were simultaneously incorporated into PVA hydrogels. The porosity, average pore size and swelling properties of the hydrogels were controlled by varying initial processing parameters, such as the relative amounts of PLGA and solvent. Average pore sizes were in the ranged 50–100 µm. The PLGA microparticles degraded within the hydrogels over time in aqueous conditions, resulting in increases in porosity and pore size. After 4 weeks in cell culture, immature cartilage tissue filled many of the pores of the hydrogels that initially contained PLGA, and proteoglycan production was proportional to the amount of PLGA. In contrast, there was little cell attachment and no proteoglycan production in control hydrogels without PLGA. The compressive moduli of the hydrogels were similar to that of healthy cartilage and increased over time from 0.05–0.1 to 0.3–0.7 MPa. The generation of a hybrid cartilage–hydrogel construct using this technique may finally allow the integration of PVA hydrogels with surrounding cartilage. Copyright

Analysis of the in vitro swelling behavior of poly(vinyl alcohol) hydrogels in osmotic pressure solution for soft tissue replacement

An osmotic solution was used to evaluate poly(vinyl alcohol) (PVA) hydrogels as potential non-degradable soft tissue replacements in vitro. Osmotic solutions are necessary in order to mimic the swelling pressure observed in vivo for soft tissues present in load-bearing joints. In vitro studies indicated that PVA hydrogels experience minimal changes in swelling with a polymer concentration of 20 wt.% PVA in phosphate-buffered saline solution (0 atm) and between 30 and 35 wt.% PVA in osmotic solution with a pressure of 0.95 atm. Swelling in osmotic pressure solutions caused decreases in the equilibrium hydrogel hydration. An investigation of hydrogel compressive modulus indicated that PVA hydrogels are within the range of articular cartilage, meniscal tissue, and the temporomandibular joint disk. Furthermore, it is possible to tailor PVA hydrogels through careful modification of the polymer concentration and freeze-thaw cycles during hydrogel preparation to match both a desired swelling ratio and a desired compressive modulus or porosity. The microstructure of the PVA hydrogels was also evaluated as a function of freeze-thaw cycles and polymer concentration to give an insight into the processes occurring during synthesis and swelling in osmotic solutions.

Synergistic Effects of SDF-1α and BMP-2 Delivery from Proteolytically Degradable Hyaluronic Acid Hydrogels for Bone Repair

In order to achieve bone repair, bone morphogenetic protein-2 (BMP-2) is typically delivered in non-physiological doses and can result in significant adverse side effects. To reduce the amount of BMP-2 necessary for bone formation, we delivered a known chemokine (stromal cell derived factor-1α, SDF-1α) in combination with BMP-2 using proteolytically degradable hydrogels. A critical-sized calvarial defect was used to determine the effect of biomolecule delivery on bone formation in vivo. The treatment group with combined SDF-1α and BMP-2 hydrogel delivery showed significantly higher bone formation when compared to hydrogels loaded with the same BMP-2 or SDF-1α concentrations alone, suggesting the combined delivery of both biomolecules synergistically improves osteogenesis.

Aging behavior of PVA hydrogels for soft tissue applications after in vitro swelling using osmotic pressure solutions.

The osmotic pressure of the medium used for in vitro swelling evaluation has been shown to have a significant effect on the swelling behavior of a material. In this study, the effect of osmotic pressure during swelling on poly(vinyl alcohol) hydrogel material properties was evaluated in vitro. Osmotic pressure solutions are necessary in order to mimic the swelling pressure observed in vivo for soft tissues present in load-bearing joints. Hydrogels were characterized after swelling by mechanical testing, X-ray diffraction and optical microscopy in the hydrated state. Results indicated that hydrogel mechanical properties remained tailorable with respect to initial processing parameters; however, significant aging occurred in osmotic solution. This was observed when evaluating the mechanical properties of the hydrogels, which, before swelling, ranged from 0.04 to 0.78 MPa but, after swelling in vitro using osmotic pressure solution, ranged from 0.32 to 0.93 MPa. Significant aging was also noted when evaluating crystallinity, with the relative crystallinity ranging between 0.4 and 5.0% before swelling and between 6.5 nd 8.0% after swelling. When compared to swelling in a non-osmotic pressure solution or in phosphate-buffered saline solution, the mechanical properties were more dependent upon the final swelling content. Furthermore, increases in crystallinity were not as significant after swelling. These results highlight the importance of choosing the appropriate swelling medium for in vitro characterization based on the desired application.

Transdermal gelation of methacrylated macromers with near-infrared light and gold nanorods

Injectable hydrogels provide locally controlled tissue bulking and a means to deliver drugs and cells to the body. The formation of hydrogels in vivo may involve the delivery of two solutions that spontaneously crosslink when mixed, with pH or temperature changes, or with light (e.g., visible or ultraviolet). With these approaches, control over the kinetics of gelation, introduction of the initiation trigger (e.g., limited penetration of ultraviolet light through tissues), or alteration of the material physical properties (e.g., mechanics) may be difficult to achieve. To overcome these limitations, we used the interaction of near-infrared (NIR) light with gold nanorods (AuNRs) to generate heat through the photothermal effect. NIR light penetrates tissues to a greater extent than other wavelengths and provides a means to indirectly initiate radical polymerization. Specifically, this heating coupled with a thermal initiator (VA-044) produced radicals that polymerized methacrylated hyaluronic acid (MeHA) and generated hydrogels. A range of VA-044 concentrations changed the gelation time, yielding a system stable at 37 ° C for 22 min that gels quickly (~3 min) when heated to 55 ° C. With a constant irradiation time (10 min) and laser power (0.3 W), different VA-044 and AuNR concentrations tuned the compressive modulus of the hydrogel. By changing the NIR irradiation time we attained a wide range of moduli at a set solution composition. In vivo mouse studies confirmed that NIR laser irradiation through tissue could gel an injected precursor solution transdermally.

Interfacial optimization of fiber-reinforced hydrogel composites for soft fibrous tissue applications

Meniscal tears are the most common orthopedic injuries to the human body, yet the current treatment of choice is a partial meniscectomy, which is known to lead to joint degeneration and osteoarthritis. As a result, there is a significant clinical need to develop materials capable of restoring function to the meniscus following an injury. Fiber-reinforced hydrogel composites are particularly suited for replicating the mechanical function of native fibrous tissues due to their ability to mimic the native anisotropic property distribution present. A critical issue with these materials, however, is the potential for the fiber-matrix interfacial properties to severely limit composite performance. In this work, the interfacial properties of an ultra-high-molecular-weight polyethylene (UHMWPE) fiber-reinforced poly(vinyl alcohol) (PVA) hydrogel are studied. A novel chemical grafting technique, confirmed using X-ray photoelectron spectroscopy, is used to improve UHMWPE-PVA interfacial adhesion. Interfacial shear strength is quantified using fiber pull-out tests. Results indicate significantly improved fiber-hydrogel interfacial adhesion after chemical grafting, where chemically grafted samples have an interfacial shear strength of 256.4±64.3kPa compared to 11.5±2.9kPa for untreated samples. Additionally, scanning electron microscopy of fiber surfaces after fiber pull-out reveal cohesive failure within the hydrogel matrix for treated fiber samples, indicating that the UHMWPE-PVA interface has been successfully optimized. Lastly, inter-fiber spacing is observed to have a significant effect on interfacial adhesion. Fibers spaced further apart have significantly higher interfacial shear strengths, which is critical to consider when optimizing composite design. The results in this study are applicable in developing similar chemical grafting techniques and optimizing fiber-matrix interfacial properties for other hydrogel-based composite systems.