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.

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.

The influence of microenvironment and extracellular matrix molecules in driving neural stem cell fate within biomaterials.

Transplantation of stem cells is a promising potential therapy for central nervous system disease and injury. The capacity for self-renewal, proliferation of progenitor cells, and multi-lineage potential underscores the need for controlling stem cell fate. Furthermore, transplantation within a hostile environment can lead to significant cell death and limited therapeutic potential. Tissue-engineered materials have been developed to both regulate stem cell fate, increase transplanted cell viability, and improve therapeutic outcomes. Traditionally, regulation of stem cell differentiation has been driven through soluble signals, such as growth factors. While these signals are important, insoluble factors from the local microenvironment or extracellular matrix (ECM) molecules also contribute to stem cell activity and fate. Understanding the microenvironment factors that influence stem cell fate, such as mechanical properties, topography, and presentation of specific ECM ligands, is necessary for designing improved biomaterials. Here we review some of the microenvironment factors that regulate stem cell fate and how they can be incorporated into biomaterials as part of potential CNS therapies.

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.

Hemostatic and Absorbent PolyHIPE-Kaolin Composites for 3D Printable Wound Dressing Materials.

A novel hemostatic and absorbent wound dressing material compatible with 3D printing is developed to address deficiencies in current wound dressing protocol. The design involves an open celled, microporous hydrogel foam via a high internal phase emulsion (HIPE) template with biocompatible components and tunable hemostatic character by kaolin loading, the viscosity and cure kinetics of which are tailored for 3D printing applications. The use of nontoxic mineral oil organic phase results in cytocompatability with human dermal fibroblasts. Kaolin distribution is shown by X-ray diffraction and elemental dispersive spectroscopy to be exfoliated and dispersed in the hydrogel dressing. In addition to demonstrating high fluid absorption and noncytotoxicity of relevant cell lines, the high internal phase emulsion polymers (polyHIPEs) also match the hemostatic performance of commercial wound dressing materials. Furthermore, the polyHIPEs display the requisite rheological properties for 3D printing that result in the fabrication of a prototype dressing with hierarchical porosity and a large number of controllable form factors.

Mechanical stabilization of proteolytically degradable polyethylene glycol dimethacrylate hydrogels through peptide interaction.

Balancing enhancement of neurite extension against loss of matrix support in synthetic hydrogels containing proteolytically degradable and bioactive signaling peptides to optimize tissue formation is difficult. Using a systematic approach, polyethylene glycol hydrogels containing concurrent continuous concentration gradients of the laminin derived bioactive signaling peptide, Ile-Lys-Val-Ala-Val (IKVAV), and collagen derived matrix metalloprotease degradable peptide, GPQGIWGQ, were fabricated and characterized. During proteolytic degradation of the concentration gradient hydrogels, the IKVAV and IWGQ cleavage fragment from GPQGIWGQ were found to interact and stabilize the bulk Young's Modulus of the hydrogel. Further testing of discrete samples containing GPQGIWGQ or its cleavage fragments, GPQG and IWGQ, indicates hydrophobic interactions between the peptides are not necessary for mechanical stabilization of the hydrogel, but changes in the concentration ratio between the peptides tethered in the hydrogel and salts and ions in the swelling solution can affect the stabilization. Encapsulation of human induced pluripotent stem cell derived neural stem cells did not reduce the mechanical properties of the hydrogel over a 14 day neural differentiation culture period, and IKVAV was found to maintain concentration dependent effects on neurite extension and mRNA gene expression of neural cytoskeletal markers, similar to previous studies. As a result, this work has significant implications for the analysis of biological studies in matrices, as the material and mechanical properties of the hydrogel may be unexpectedly temporally changing during culture due to interactions between peptide signaling elements, underscoring the need for greater matrix characterization during the degradation and cell culture. STATEMENT OF SIGNIFICANCE: Greater emulation of the native extracellular matrix is necessary for tissue formation. To achieve this, matrices are becoming more complex, often including multiple bioactive signaling elements. However, peptide signaling in polyethylene glycol matrices and amino acids interactions between peptides can affect hydrogel material and mechanical properties, but are rarely studied. The current study identifies such an interaction between laminin derived peptide, IKVAV, and collagen derived matrix metalloprotease degradable peptide, GPQGIWGQ. Previous studies using these peptides did not identify their interactions' ability to mechanically stabilize the hydrogel during degradation. This work underscores the need for greater matrix characterization and consideration of bioactive signaling element effects temporally on the matrix's material and mechanical properties, as they can contribute to cellular response.

Winner of the society for biomaterials student award in the Ph.D. category for the annual meeting of the society for biomaterials, april 11-14, 2018, Atlanta, GA: Development of a bimodal, in situ crosslinking method to achieve multifactor release from electrospun gelatin.

To better mimic native tissue microenvironments, current efforts have moved beyond single growth factor delivery to more complex multiple growth factor delivery with distinct release profiles. Electrospun gelatin, a widely investigated drug delivery vehicle, requires postprocessing crosslinking techniques that generate a mesh with uniform crosslinking density, limiting the ability to deliver multiple factors at different rates. Herein, we describe a method to independently control release of multiple factors from a single electrospun gelatin mesh. Two in situ crosslinking modalities, photocrosslinking of methacyrlated gelatin and reactive crosslinking of gelatin with a diisocyanate, are coelectrospun to generate distinct fiber populations with different crosslinking chemistry and density in a single mesh. The photocrosslinked gelatin-methacrylate resulted in a relatively rapid release of a model protein (48 ± 12% at day 1, 96 ± 3% at day 10) due to diffusion of embedded protein from the crosslinked fibers. The reactive crosslinking system displayed a more sustained release (7 ± 5% at day 1, 33 ± 2% at day 10) that was attributed to the conjugation of protein to gelatin with the diisocyanate, requiring degradation of gelatin prior to diffusion out of the fibers. Both modalities displayed tunable release profiles. Subsequent release studies of a cospun mesh with two different crosslinked fiber populations confirmed that the cospun mesh displayed multifactor release with independent release profiles. Overall, this bimodal, in situ crosslinking approach enables the delivery of multiple factors with distinct release kinetics from a single mesh and is expected to have broad utility in tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1155-1164, 2018.

Effects of free radical initiators on polyethylene glycol dimethacrylate hydrogel properties and biocompatibility.

Many studies have utilized Irgacure 2959 photopolymerized poly(ethylene glycol) (PEG) hydrogels for tissue engineering application development. Due to the limited penetration of ultraviolet light through tissue, Irgacure 2959 polymerized hydrogels are not suitable for use in tissues where material injection is desirable, such as the spinal cord. To address this, several free radical initiators (thermal initiator VA044, ammonium persulfate (APS)/TEMED reduction-oxidation reaction, and Fenton chemistry) are evaluated for their effects on the material and mechanical properties of PEG hydrogels compared with Irgacure 2959. To emulate the effects of endogenous thiols on in vivo polymerization, the effects of chain transfer agent (CTA) dithiothreitol on gelation rates, material properties, Young's and shear modulus, are examined. Mouse embryonic stem cells and human induced pluripotent stem cell derived neural stem cells were used to investigate the cytocompatibility of each polymerization. VA044 and Fenton chemistry polymerization of PEG hydrogels both had gelation rates and mechanical properties that were highly susceptible to changes in CTA concentration and showed poor cytocompatibility. APS/TEMED polymerized hydrogels maintained consistent gelation rates and mechanical properties at high CTA concentration and had a similar cytocompatibility as Irgacure 2959 when cells were encapsulated within the PEG hydrogels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3059-3068, 2017.

Human Induced Pluripotent Stem Cell Derived Neural Stem Cell Survival and Neural Differentiation on Polyethylene Glycol Dimethacrylate Hydrogels Containing a Continuous Concentration Gradient of N-Cadherin Derived Peptide His-Ala-Val-Asp-Ile.

Although preclinical models of spinal cord injury have shown that matrix inclusion in stem cell therapy leads to greater neurological improvements than that including cells alone, there has been insufficient matrix optimization for human cells. N-Cadherin influences the development and maintenance of neural tissue, but the effects of N-cadherin derived peptide His-Ala-Val-Asp-Ile (HAVDI) on the survival, neurite extension, and expression of neural differentiation markers in human induced pluripotent stem cell derived neural stems (hNSC) have not been widely examined. Using polyethylene glycol hydrogels containing a continuous gradient of HAVDI, this study identifies concentration dependent effects on hNSC survival and neural differentiation.

Synthesis and Characterization of Plug-and-Play Polyurethane Urea Elastomers as Biodegradable Matrixes for Tissue Engineering Applications.

The highly tunable mechanical properties and resilience of polyurethanes make them promising candidates for tissue engineering applications. Biodegradability is conferred by incorporation of hydrolytically or enzymatically cleavable moieties into the polyurethane structure. A common choice for the biodegradable soft segment is a poly(ether ester) triblock copolymer synthesized by ring opening polymerization of the polyester from a polyether macroinitiator. Herein, we describe a new "plug-and-play" approach for triblock synthesis based on urethane block coupling that enables finer control of block lengths and ease of segmental tuning. The inclusion of urethane linkages in the soft segment was also hypothesized to promote hydrogen bonding between the segments with an associated increase in modulus, tensile strength, and ultimate elongation. Hard segment content of the biodegradable polyurethane urea was varied to demonstrate the tunable tensile properties and degradation rate. As expected, increasing hard segment content led to large increases in initial secant modulus and tensile strength. A corollary decrease in ultimate elongation, elastic recovery, and degradation rate was also observed with increasing hard segment content. Finally, cytocompatibility and hydrolytic degradation of electrospun polyurethane meshes were evaluated to establish the potential use of these biodegradable matrixes as tissue engineering scaffolds. All of the polyurethane formulations displayed comparable cytocompatibilty to tissue culture plastic controls and hydrolytic chain scission of the polyester soft segment. Overall, this synthetic approach provides a platform to produce biodegradable polyurethane ureas with enhanced control over segmental chemistry, mechanical properties, and degradation rate.

Stem cells for spinal cord injury: Strategies to inform differentiation and transplantation.

The complex pathology of spinal cord injury (SCI), involving a cascade of secondary events and the formation of inhibitory barriers, hampers regeneration across the lesion site and often results in irreversible loss of motor function. The limited regenerative capacity of endogenous cells after SCI has led to a focus on the development of cell therapies that can confer both neuroprotective and neuroregenerative benefits. Stem cells have emerged as a candidate cell source because of their ability to self-renew and differentiate into a multitude of specialized cell types. While ethical and safety concerns impeded the use of stem cells in the past, advances in isolation and differentiation methods have largely mitigated these issues. A confluence of work in stem cell biology, genetics, and developmental neurobiology has informed the directed differentiation of specific spinal cell types. After transplantation, these stem cell-derived populations can replace lost cells, provide trophic support, remyelinate surviving axons, and form relay circuits that contribute to functional recovery. Further refinement of stem cell differentiation and transplantation methods, including combinatorial strategies that involve biomaterial scaffolds and drug delivery, is critical as stem cell-based treatments enter clinical trials. Biotechnol. Bioeng. 2017;114: 245-259. © 2016 Wiley Periodicals, Inc.

Response to di-functionalized hyaluronic acid with orthogonal chemistry grafting at independent modification sites in rodent models of neural differentiation and spinal cord injury.

Hyaluronic acid (HA) with one reactive moiety grafted to the backbone is a commonly used matrix in tissue engineering. The addition of a second orthogonal moiety to the backbone allows for greater control in bioactive signal tethering and gelation. In this study, thiol and azide functional groups were grafted to the HA backbone at separate modification sites. NMR, FT-IR, colorimetric assay, and radio-TLC activity were used to confirm and quantify thiol and azide grafting to the HA backbone. Various ratios of di-functional HA (dif HA) and methacrylate HA (mHA) were used to encapsulate mouse embryonic stem cells in order to examine the neural differentiation of the cells. Greater neural maturation was observed in hydrogels containing a higher percentage of dif HA compared to mHA over a six day neural differentiation time course. This formulation was then tested in a contusion spinal cord injury model for biological effect and was found to reduce the ED1+ area in the spinal cord compared to control and allow for host axon extension into the matrix filled lesion area. These results indicate that dif HA is supportive of neural differentiation and can reduce inflammation without additional bioactive signal tethering. dif HA is a promising matrix base for the central nervous system, which should be further developed.

Combination therapy of stem cell derived neural progenitors and drug delivery of anti-inhibitory molecules for spinal cord injury.

Regeneration of lost synaptic connections following spinal cord injury (SCI) is limited by local ischemia, cell death, and an excitotoxic environment, which leads to the development of an inhibitory glial scar surrounding a cystic cavity. While a variety of single therapy interventions provide incremental improvements to functional recovery after SCI, they are limited; a multifactorial approach that combines several single therapies may provide a better chance of overcoming the multitude of obstacles to recovery. To this end, fibrin scaffolds were modified to provide sustained delivery of neurotrophic factors and anti-inhibitory molecules, as well as encapsulation of embryonic stem cell-derived progenitor motor neurons (pMNs). In vitro characterization of this combination scaffold confirmed that pMN viability was unaffected by culture alongside sustained delivery systems. When transplanted into a rat sub-acute SCI model, fibrin scaffolds containing growth factors (GFs), anti-inhibitory molecules without pMNs, or pMNs with GFs had lower chondroitin sulfate proteoglycan levels compared to scaffolds containing anti-inhibitory molecules with pMNs. Scaffolds containing pMNs, but not anti-inhibitory molecules, showed survival, differentiation into neuronal cell types, axonal extension in the transplant area, and the ability to integrate into host tissue. However, the combination of pMNs with sustained-delivery of anti-inhibitory molecules led to reduced cell survival and increased macrophage infiltration. While combination therapies retain potential for effective treatment of SCI, further work is needed to improve cell survival and to limit inflammation. STATEMENT OF SIGNIFICANCE: Spinal cord injury (SCI) creates a highly complex inhibitory environment with a multitude of obstacles that limit recovery. Many therapeutic options have been developed to overcome single obstacles, but single therapies typically only lead to limited functional improvement. Therefore combination therapies may improve recovery by targeting several inhibitory obstacles simultaneously. The present study used biomaterial scaffolds to combine the sustained release of anti-inhibitory molecules and growth factors with cell transplantation of highly purified progenitor motor neurons. This expands upon previously established biomaterial scaffolds by supporting surviving cells, limiting inhibition from the extracellular environment, and replenishing lost cell populations. We show that while promising, certain combinations may exacerbate negative side-effects instead of augmenting positive features.

Sustained dual drug delivery of anti-inhibitory molecules for treatment of spinal cord injury.

Myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs) are major contributors to axon growth inhibition following spinal cord injury and limit functional recovery. The NEP1-40 peptide competitively binds the Nogo receptor and partially blocks inhibition from MAIs, while chondroitinase ABC (ChABC) enzymatically digests CSPGs, which are upregulated at the site of injury. In vitro studies showed that the combination of ChABC and NEP1-40 increased neurite extension compared to either treatment alone when dissociated embryonic dorsal root ganglia were seeded onto inhibitory substrates containing both MAIs and CSPGs. Furthermore, the ability to provide sustained delivery of biologically active ChABC and NEP1-40 from biomaterial scaffolds was achieved by loading ChABC into lipid microtubes and NEP1-40 into poly (lactic-co-glycolic acid) (PLGA) microspheres, obviating the need for invasive intrathecal pumps or catheters. Fibrin scaffolds embedded with the drug delivery systems (PLGA microspheres and lipid microtubes) were capable of releasing active ChABC for up to one week and active NEP1-40 for over two weeks in vitro. In addition, the loaded drug delivery systems in fibrin scaffolds decreased CSPG deposition and development of a glial scar, while also increasing axon growth after spinal cord injury in vivo. Therefore, the sustained, local delivery of ChABC and NEP1-40 within the injured spinal cord may block both myelin and CSPG-associated inhibition and allow for improved axon growth.