Injectable polymerized high internal phase emulsions with rapid in situ curing.

Polymerized high internal phase emulsions (polyHIPEs) have been utilized in the creation of injectable scaffolds that cure in situ to fill irregular bone defects and potentially improve tissue healing. Previously, thermally initiated scaffolds required hours to cure, which diminished the potential for clinical translation. Here, a double-barrel syringe system for fabricating redox-initiated polyHIPEs with dramatically shortened cure times upon injection was demonstrated with three methacrylated macromers. The polyHIPE cure time, compressive properties, and pore architecture were investigated with respect to redox initiator chemistry and concentration. Increased concentrations of redox initiators reduced cure times from hours to minutes and increased the compressive modulus and strength without compromising the pore architecture. Additionally, storage of the uncured emulsion at reduced temperatures for 6 months was shown to have minimal effects on the resulting graft properties. These studies indicate that the uncured emulsions can be stored in the clinic until they are needed and then rapidly cured after injection to rigid, high-porosity scaffolds. In summary, we have improved upon current methods of generating injectable polyHIPE grafts to meet translational design goals of long storage times and rapid curing (<15 min) without sacrificing porosity or mechanical properties.

Solvent-free fabrication of polyHIPE microspheres for controlled release of growth factors.

The growth factor bone morphogenetic protein 2 (BMP-2) is utilized in surgical procedures to improve bone regeneration; however, current treatments deliver BMP-2 at amounts greater than 100,000 fold of physiological levels, which increases treatment costs and risk of side effects. Drug-eluting microcarriers developed to improve these therapies have faced significant commercialization challenges including particle size distributions, solvent removal, low encapsulation efficiency, and bioactivity loss. In this study, a solvent-free method is presented for fabrication of uniform polyHIPE microspheres for controlled growth factor release. Emulsion templating principles and fluid dynamics were used to fabricate uniform particles with tunable particle size (200-800 µm) and pore size (10-30 µm). The ability to independently tune particle and pore size is expected to provide excellent control of release kinetics. Overall, this solvent-free method for making porous microspheres displays strong promise for the controlled release of BMP-2 and other growth factors.

Preclinical evaluation of a commercially available biofilm disrupting wound lavage for musculoskeletal trauma.

BACKGROUND: Treatment of open fractures remains a significant challenge in trauma care as these fractures are accompanied by extensive soft tissue damage, exposing the wound site to contaminants and increasing infection risk. Formation of biofilm, a capsule-like environment that acts as a barrier to treatment, is a primary mode by which infecting pathogens persist at the wound site. Therefore, a pressing need exists to identify irrigation methods that can disrupt biofilm and expose pathogens to treatment. This study aims to evaluate the antibiofilm wound lavage, Bactisure™, in comparison with saline for care of severe musculoskeletal wounds and elucidate potential effects on antibiotic treatment success. METHODS: UAMS-1 Staphylococcus aureus biofilms were formed in vitro and treated with Bactisure™ wound lavage or sterile normal saline, alone, or in combination with sub-biofilm inhibitory levels of vancomycin. Characterization methods included quantification of biofilm biomass, quantification of viable biofilm bacteria, and biofilm matrix imaging. For in vivo assessment, a delayed treatment model of contaminated open fracture was used wherein a critical-sized defect was created in a rat femur and wound site inoculated with UAMS-1. Following a 6 h delay, wounds were debrided, irrigated with lavage of interest, and antibiotic treatments administered. Bacterial enumeration was performed on bone and hardware samples after two weeks. RESULTS: An immediate reduction in biofilm biomass was observed in vitro following antibiofilm lavage treatment, with a subsequent 2- to 3- log reduction in viable bacteria achieved after 24 h. Furthermore, biofilms treated with antibiofilm lavage in combination with vancomycin exhibited a minor, but statistically significant, decrease in viable bacteria compared to irrigation alone. In vivo, a minor, not statistically significant, decrease in median bioburden was observed for the antibiofilm lavage compared to saline when used in combination with antibiotics. However, the percentage of bone and hardware samples with detectable bacteria was reduced from 50 to 38%. CONCLUSIONS: These results suggest that the antibiofilm wound lavage, Bactisure™, may hold promise in mitigating infection in contaminated musculoskeletal wounds and warrants further investigation. Here, we proposed multiple mechanisms in vitro by which this antibiofilm lavage may help mitigate infection, and demonstrate this treatment slightly outperforms saline in controlling bioburden in vivo.

Administration of a selective retinoic acid receptor-γ agonist improves neuromuscular strength in a rodent model of volumetric muscle loss.

PURPOSE: Volumetric muscle loss is a uniquely challenging pathology that results in irrecoverable functional deficits. Furthermore, a breakthrough drug or bioactive factor has yet to be established that adequately improves repair of these severe skeletal muscle injuries. This study sought to assess the ability of an orally administered selective retinoic acid receptor-γ agonist, palovarotene, to improve recovery of neuromuscular strength in a rat model of volumetric muscle loss. METHODS: An irrecoverable, full thickness defect was created in the tibialis anterior muscle of Lewis rats and animals were survived for 4 weeks. Functional recovery of the tibialis anterior muscle was assessed in vivo via neural stimulation and determination of peak isometric torque. Histological staining was performed to qualitatively assess fibrous scarring of the defect site. RESULTS: Treatment with the selective retinoic acid receptor-γ agonist, palovarotene, resulted in a 38% improvement of peak isometric torque in volumetric muscle loss affected limbs after 4 weeks of healing compared to untreated controls. Additionally, preliminary histological assessment suggests that oral administration of palovarotene reduced fibrous scarring at the defect site. CONCLUSIONS: These results highlight the potential role of selective retinoic acid receptor-γ agonists in the design of regenerative medicine platforms to maximize skeletal muscle healing. Additional studies are needed to further elucidate cellular responses, optimize therapeutic delivery, and characterize synergistic potential with adjunct therapies.

Comparative efficacy of resorbable fiber wraps loaded with gentamicin sulfate or gallium maltolate in the treatment of osteomyelitis.

The high incidence of osteomyelitis associated with critical-sized bone defects raises clinical challenges in fracture healing. Clinical use of antibiotic-loaded bone cement as an adjunct therapy is limited by incompatibility with many antimicrobials, sub-optimal release kinetics, and requirement of surgical removal. Furthermore, overuse of antibiotics can lead to bacterial modifications that increase efflux, decrease binding, or cause inactivation of the antibiotics. Herein, we compared the efficacy of gallium maltolate, a new metal-based antimicrobial, to gentamicin sulfate released from electrospun poly(lactic-co-glycolic) acid (PLGA) wraps in the treatment of osteomyelitis. In vitro evaluation demonstrated sustained release of each antimicrobial up to 14 days. A Kirby Bauer assay indicated that the gentamicin sulfate-loaded wrap inhibited the growth of osteomyelitis-derived isolates, comparable to the gentamicin sulfate powder control. In contrast, the gallium maltolate-loaded wrap did not inhibit bacteria growth. Subsequent microdilution assays indicated a lower than expected sensitivity of the osteomyelitis strain to the gallium maltolate with release concentrations below the threshold for bactericidal activity. A comparison of the selectivity indices indicated that gentamicin sulfate was less toxic and more efficacious than gallium maltolate. A pilot study in a contaminated femoral defect model confirmed that the sustained release of gentamicin sulfate from the electrospun wrap resulted in bacteria density reduction on the surrounding bone, muscle, and hardware below the threshold that impedes healing. Overall, these findings demonstrate the efficacy of a resorbable, antimicrobial wrap that can be used as an adjunct or stand-alone therapy for controlled release of antimicrobials in the treatment of osteomyelitis.

In Vivo Characterization of Poly(ethylene glycol) Hydrogels with Thio-ß Esters.

Resorbable hydrogels have numerous potential applications in tissue engineering and drug delivery due to their highly tunable properties and soft tissue-like mechanical properties. The incorporation of esters into the backbone of poly(ethylene glycol) hydrogels has been used to develop libraries of hydrogels with tunable degradation rates. However, these synthetic strategies used to increase degradation rate often result in undesired changes in the hydrogel physical properties such as matrix modulus or swelling. In an effort to decouple degradation rate from other hydrogel properties, we inserted thio-ß esters into the poly(ethylene glycol)-diacrylate backbone to introduce labile bonds without changing macromer molecular weight. This allowed the number of hydrolytically labile thio-ß esters to be controlled through changing the ratios of this modified macromer to the original macromer without affecting network properties. The retention of hydrogel properties at different macromer ratios was confirmed by measuring gel fraction, swelling ratio, and compressive modulus. The tunable degradation profiles were characterized both in vitro and in vivo. Following confirmation of cytocompatibility after exposure to the hydrogel degradation products, the in vivo host response was evaluated in comparison to medical grade silicone. Collectively, this work demonstrates the utility and tunability of these hydrolytically degradable hydrogels for a wide variety of tissue engineering applications.

Porous PolyHIPE microspheres for protein delivery from an injectable bone graft.

Delivery of osteoinductive factors such as bone morphogenetic protein 2 (BMP-2) has emerged as a prominent strategy to improve regeneration in bone grafting procedures. However, it remains challenging to identify a carrier that provides the requisite loading efficiency and release kinetics without compromising the mechanical properties of the bone graft. Previously, we reported on porous, polymerized high internal phase emulsion (polyHIPE) microspheres fabricated using controlled fluidics. Uniquely, this solvent-free method provides advantages over current microsphere fabrication strategies including in-line loading of growth factors to improve loading efficiency. In the current study, we utilized this platform to fabricate protein-loaded microspheres and investigated the effect of particle size (∼400 vs ∼800 µm) and pore size (∼15 vs 30 µm) on release profiles. Although there was no significant effect of these variables on the substantial burst release profile of the microspheres, the incorporation of the protein-loaded microspheres within the injectable polyHIPE resulted in a sustained release of protein from the bulk scaffold over a two-week period with minimal burst release. Bioactivity retention of encapsulated BMP-2 was confirmed first using a genetically-modified osteoblast reporter cell line. A functional assay with human mesenchymal stem cells established that the BMP-2 release from microspheres induced osteogenic differentiation. Finally, microsphere incorporation had minimal effect on the cure and compressive properties of an injectable polyHIPE bone graft. Overall, this work demonstrates that these microsphere-polyHIPE composites have strong potential to enhance bone regeneration through controlled release of BMP-2 and other growth factors. STATEMENT OF SIGNIFICANCE: This manuscript describes a method for solvent-free fabrication of porous microspheres from high internal phase emulsions using a controlled fluids setup. The principles of emulsion templating and fluid dynamics provide exceptional control of particle size and pore architecture. In addition to the advantage of solvent-free fabrication, this method provides in-line loading of protein directly into the pores of the microspheres with high loading efficiencies. The incorporation of the protein-loaded microspheres within an injectable polyHIPE scaffold resulted in a sustained release of protein over a two-week period with minimal burst release. Retention of BMP-2 bioactivity and incorporation of microspheres with minimal effect on scaffold compressive properties highlights the potential of these new bone grafts.

Bactericidal activity of 3D-printed hydrogel dressing loaded with gallium maltolate.

Chronic wounds are projected to reach epidemic proportions worldwide because of the aging population and the increasing incidence of diabetes. Despite extensive research, infection remains one of the leading sources of complications in chronic wounds, resulting in improper healing, biofilm formation, and lower extremity amputation. To address the limitations of standard treatments, we have developed a hydrogel wound dressing with self-tuning moisture control that incorporates a novel antimicrobial agent to eliminate and prevent infection. 3D-printing of a hydrogel dressing with dual porosity resulted in a new dressing with greater flexibility, increased water uptake, and more rapid swelling than bulk hydrogel dressings. Additionally, gallium maltolate (GaM) was incorporated into the dressing to investigate the efficacy of this antimicrobial agent. Loading profiles, release kinetics, and the bactericidal activity against Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus) of GaM were investigated in vitro to identify target profiles that supported infection control. Finally, GaM-loaded hydrogel dressings were evaluated in vivo, utilizing a murine splinted-wound model that was inoculated with S. aureus. In comparison to an untreated control, GaM dressings markedly reduced the wound bacterial load without compromising wound closure rates. Overall, this work demonstrates the utility of a 3D-printed hydrogel dressing as an antimicrobial dressing to control infection in chronic wounds.

Improved in situ seeding of 3D printed scaffolds using cell-releasing hydrogels.

The design of tissue engineered scaffolds based on polymerized high internal phase emulsions (polyHIPEs) has emerged as a promising bone grafting strategy. We previously reported the ability to 3D print emulsion inks to better mimic the structure and mechanical properties of native bone while precisely matching defect geometry. In the current study, redox-initiated hydrogel carriers were investigated for in situ delivery of human mesenchymal stem cells (hMSCs) utilizing the biodegradable macromer, poly(ethylene glycol)-dithiothreitol. Hydrogel carrier properties including network formation time, sol-gel fraction, and swelling ratio were modulated to achieve rapid cure without external stimuli and a target cell-release period of 5-7 days. These in situ carriers enabled improved distribution of hMSCs in 3D printed polyHIPE grafts over standard suspension seeding. Additionally, carrier-loaded polyHIPEs supported sustained cell viability and osteogenic differentiation of hMSCs post-release. In summary, these findings demonstrate the potential of this in situ curing hydrogel carrier to enhance the cell distribution and retention of hMSCs in bone grafts. Although initially focused on improving bone regeneration, the ability to encapsulate cells in a hydrogel carrier without relying on external stimuli that can be attenuated in large grafts or tissues is expected to have a wide range of applications in tissue engineering.

Fabrication of biomimetic bone grafts with multi-material 3D printing.

Extrusion deposition is a versatile method for the 3D printing of biomaterials such as hydrogels, ceramics, and suspensions. Recently, a new class of emulsion inks were developed that can be used to create tunable, hierarchically porous materials with a cure-on-dispense method. Propylene fumarate dimethacrylate (PFDMA) was selected to fabricate bone grafts using this technology due to its established biocompatibility, osteoconductivity, and good compressive properties. Scaffolds fabricated from PFDMA emulsion inks displayed compressive modulus and yield strength of approximately 15 and 1 MPa, respectively. A decrease in infill (from 100% to 70%) resulted in a six-fold increase in permeability; however, there was also a corollary decrease in mechanical properties. In order to generate scaffolds with increased permeability without sacrificing mechanical strength, a biomimetic approach to scaffold design was used to reinforce the highly porous emulsion inks with a dense cortical shell of thermoplastic polyester. Herein, we present an open source method for printing multi-material bone grafts based on PFDMA polyHIPEs with hierarchical porosity and reinforced with a dense shell of poly(ε-caprolactone) (PCL) or poly(lactic acid) (PLA). A multi-modal printing setup was first developed that combined paste extrusion and high temperature thermoplastic extrusion with high positional accuracy in dual deposition. Scaffolds printed with a PCL shell displayed compressive modulus and yield strength of approximately 30 and 3 MPa, respectively. Scaffolds printed with a PLA shell showed compressive modulus and yield strength of approximately 100 and 10 MPa, respectively. By combining this new paste extrusion of emulsion inks with traditional thermoplastic extrusion printing, we have created scaffolds with superior strength that promote cell viability and proliferation of human mesenchymal stem cells. The development of this technique shows great promise for the fabrication of a myriad of other complex tissue engineered scaffolds.

Prevention of Oxygen Inhibition of PolyHIPE Radical Polymerization using a Thiol-based Crosslinker.

Polymerized high internal phase emulsions (polyHIPEs) are highly porous constructs currently under investigation as tissue engineered scaffolds. We previously reported on the potential of redox-initiated polyHIPEs as injectable bone grafts that space fill irregular defects with improved integration and rapid cure. Upon subsequent investigation, the radical-initiated cure of these systems rendered them susceptible to oxygen inhibition with an associated increase in uncured macromer in the clinical setting. In the current study, polyHIPEs with increased resistance to oxygen inhibition were fabricated utilizing a tetrafunctional thiol, pentaerythritol tetrakis(3-mercaptoproprionate), and the biodegradable macromer, propylene fumarate dimethacrylate. Increased concentrations of the tetrathiol additive provided improved oxygen resistance as confirmed by polyHIPE gel fraction while retaining the requisite rapid cure rate, compressive properties, and pore architecture for use as an injectable bone graft. Additionally, thiol-methacrylate polyHIPEs exhibited increased degradation under accelerated conditions and supported critical markers of human mesenchymal stem cell activity. In summary, we have improved upon current methods of fabricating injectable polyHIPE grafts to meet translational design goals of improved polymerization kinetics under clinically relevant conditions without sacrificing key scaffold properties.

Osteoinductive PolyHIPE Foams as Injectable Bone Grafts.

We have recently fabricated biodegradable polyHIPEs as injectable bone grafts and characterized the mechanical properties, pore architecture, and cure rates. In this study, calcium phosphate nanoparticles and demineralized bone matrix (DBM) particles were incorporated into injectable polyHIPE foams to promote osteoblastic differentiation of mesenchymal stem cells (MSCs). Upon incorporation of each type of particle, stable monoliths were formed with compressive properties comparable to control polyHIPEs. Pore size quantification indicated a negligible effect of all particles on emulsion stability and resulting pore architecture. Alizarin red calcium staining illustrated the incorporation of calcium phosphate particles at the pore surface, while picrosirius red collagen staining illustrated collagen-rich DBM particles within the monoliths. Osteoinductive particles had a negligible effect on the compressive modulus (∼30 MPa), which remained comparable to human cancellous bone values. All polyHIPE compositions promoted human MSC viability (∼90%) through 2 weeks. Furthermore, gene expression analysis indicated the ability of all polyHIPE compositions to promote osteogenic differentiation through the upregulation of bone-specific markers compared to a time zero control. These findings illustrate the potential for these osteoinductive polyHIPEs to promote osteogenesis and validate future in vivo evaluation. Overall, this work demonstrates the ability to incorporate a range of bioactive components into propylene fumarate dimethacrylate-based injectable polyHIPEs to increase cellular interactions and direct specific behavior without compromising scaffold architecture and resulting properties for various tissue engineering applications.