Utilizing Robust Design to Optimize Composite Bioadhesive for Promoting Dermal Wound Repair.

Catechol-modified bioadhesives generate hydrogen peroxide (H2O2) during the process of curing. A robust design experiment was utilized to tune the H2O2 release profile and adhesive performance of a catechol-modified polyethylene glycol (PEG) containing silica particles (SiP). An L9 orthogonal array was used to determine the relative contributions of four factors (the PEG architecture, PEG concentration, phosphate-buffered saline (PBS) concentration, and SiP concentration) at three factor levels to the performance of the composite adhesive. The PEG architecture and SiP wt% contributed the most to the variation in the results associated with the H2O2 release profile, as both factors affected the crosslinking of the adhesive matrix and SiP actively degraded the H2O2. The predicted values from this robust design experiment were used to select the adhesive formulations that released 40-80 µM of H2O2 and evaluate their ability to promote wound healing in a full-thickness murine dermal wound model. The treatment with the composite adhesive drastically increased the rate of the wound healing when compared to the untreated controls, while minimizing the epidermal hyperplasia. The release of H2O2 from the catechol and soluble silica from the SiP contributed to the recruitment of keratinocytes to the wound site and effectively promoted the wound healing.

Thermomagnetic-Responsive Self-Folding Microgrippers for Improving Minimally Invasive Surgical Techniques and Biopsies.

Traditional open surgery complications are typically due to trauma caused by accessing the procedural site rather than the procedure itself. Minimally invasive surgery allows for fewer complications as microdevices operate through small incisions or natural orifices. However, current minimally invasive tools typically have restricted maneuverability, accessibility, and positional control of microdevices. Thermomagnetic-responsive microgrippers are microscopic multi-fingered devices that respond to temperature changes due to the presence of thermal-responsive polymers. Polymeric devices, made of poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-AAc) and polypropylene fumarate (PPF), self-fold due to swelling and contracting of the hydrogel layer. In comparison, soft metallic devices feature a pre-stressed metal bilayer and polymer hinges that soften with increased temperature. Both types of microdevices can self-actuate when exposed to the elevated temperature of a cancerous tumor region, allowing for direct targeting for biopsies. Microgrippers can also be doped to become magnetically responsive, allowing for direction without tethers and the retrieval of microdevices containing excised tissue. The smaller size of stimuli-responsive microgrippers allows for their movement through hard-to-reach areas within the body and the successful extraction of intact cells, RNA and DNA. This review discusses the mechanisms of thermal- and magnetic-responsive microdevices and recent advances in microgripper technology to improve minimally invasive surgical techniques.

Electroactive Polymeric Composites to Mimic the Electromechanical Properties of Myocardium in Cardiac Tissue Repair.

Due to the limited regenerative capabilities of cardiomyocytes, incidents of myocardial infarction can cause permanent damage to native myocardium through the formation of acellular, non-conductive scar tissue during wound repair. The generation of scar tissue in the myocardium compromises the biomechanical and electrical properties of the heart which can lead to further cardiac problems including heart failure. Currently, patients suffering from cardiac failure due to scarring undergo transplantation but limited donor availability and complications (i.e., rejection or infectious pathogens) exclude many individuals from successful transplant. Polymeric tissue engineering scaffolds provide an alternative approach to restore normal myocardium structure and function after damage by acting as a provisional matrix to support cell attachment, infiltration and stem cell delivery. However, issues associated with mechanical property mismatch and the limited electrical conductivity of these constructs when compared to native myocardium reduces their clinical applicability. Therefore, composite polymeric scaffolds with conductive reinforcement components (i.e., metal, carbon, or conductive polymers) provide tunable mechanical and electroactive properties to mimic the structure and function of natural myocardium in force transmission and electrical stimulation. This review summarizes recent advancements in the design, synthesis, and implementation of electroactive polymeric composites to better match the biomechanical and electrical properties of myocardial tissue.

Controlling the Release of Hydrogen Peroxide from Catechol-Based Adhesive Using Silica Nanoparticle.

Catechol-based bioadhesives generate hydrogen peroxide (H2O2) as a byproduct during the curing process. H2O2 can have both beneficial and deleterious effects on biological systems depending on its concentration. To control the amount of H2O2 released from catechol-containing polyethylene glycol-based adhesive (PEG-DA), adhesive was formulated with silica nanoparticles (SiNP) prepared with increased porosity and acid treatment to increase Si-OH surface content. These SiNP demonstrated increased surface area, which promoted interaction with catechol and resulted in increased cure rate, bulk mechanical properties and adhesive properties of PEG-DA. Most importantly, SiNP demonstrated a 50% reduction in the released H2O2 while improving the cell viability and proliferation of three primary cell types, including rat dermal fibroblasts, human epidermal keratinocytes, and human tenocytes. Additionally, SiNP degraded into soluble Si, which also contributed to increased cell proliferation. Incorporation of porous and acid-treated SiNP can be a useful approach to simultaneously modulate the concentration of H2O2 while increasing the adhesive performance of catechol-based adhesives.

Hydroxyl Radical Generation Through the Fenton-Like Reaction of Hematin- and Catechol-Functionalized Microgels.

Hydroxyl radical (•OH) is a potent reactive oxygen species with the ability to degrade hazardous organic compounds, kill bacteria, and inactivate viruses. However, an off-the-shelf, portable, and easily activated biomaterial for generating •OH does not exist. Here, microgels were functionalized with catechol, an adhesive moiety found in mussel adhesive proteins, and hematin (HEM), a hydroxylated Fe3+ ion-containing porphyrin derivative. When the microgel was hydrated in an aqueous solution with physiological pH, molecular oxygen in the solution oxidized catechol to generate H2O2, which was further converted to •OH by HEM. The generated •OH was able to degrade organic dyes, including orange II and malachite green. Additionally, the generated •OH was antimicrobial against both gram-negative (Escherichia coli) and gram-positive (Staphylococcus epidermidis) bacteria with the initial concentration of 106-107 CFU/mL. These microgels also reduced the infectivity of a non-enveloped porcine parvovirus and an enveloped bovine viral diarrhea virus by 3.5 and 4.5 log reduction values, respectively (99.97-99.997% reduction in infectivity). These microgels were also functionalized with positively charged [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METAC), which significantly enhanced the antibacterial and antiviral activities through electrostatic interaction between the negatively charged pathogens and the microgel. These microgels can potentially serve as a lightweight and portable source of disinfectant, for an on-demand generation of •OH with a wide range of applications.

Development of an Injectable Nitric Oxide Releasing Poly(ethylene) Glycol-Fibrin Adhesive Hydrogel.

Fibrin microparticles were incorporated into poly(ethylene) glycol (PEG)-fibrinogen hydrogels to create an injectable, composite that could serve as a wound healing support and vehicle to deliver therapeutic factors for tissue engineering. Nitric oxide (NO), a therapeutic agent in wound healing, was loaded into fibrin microparticles by blending S-Nitroso-N-acetyl penicillamine (SNAP) with a fibrinogen solution. The incorporation of microparticles affected swelling behavior and improved tissue adhesivity of composite hydrogels. Controlled NO release was induced via photolytic and thermal activation, and modulated by weight percent of particles incorporated. These NO-releasing composites were non-cytotoxic in culture. Cells maintained morphology, viability, and proliferative character. Fibrin microparticles loaded with SNAP and incorporated into a PEG-fibrinogen matrix, creates a novel injectable composite hydrogel that offers improved tissue adhesivity and inducible NO-release for use as a regenerative support for wound healing and tissue engineering applications.

Multifunctional Biomedical Adhesives.

Currently available biomedical adhesives are mainly engineered to have one function (i.e., providing mechanical support for the repaired tissue). To improve the performance of existing bioadhesives and broaden their applications in medicine, numerous multifunctional bioadhesives are reported in the literature. These adhesives can be categorized as passive or active by design. Passive multifunctional bioadhesives contain inherent compositions and structural designs that can carry out additional functions without added external influences. These adhesives exhibit new functionalities such as antimicrobial properties, self-healing abilities, the ability to promote cellular ingrowth, and the ability to be reshaped. Conversely, active multifunctional bioadhesives respond to environmental changes (e.g., pH, temperature, electricity, light, and biomolecule concentration), which initiate a change in the adhesive to release encapsulated drugs or to activate or deactivate the bioadhesive for interfacial binding. This review article highlights recent advances in multifunctional bioadhesives.

Control of cellular adhesion and myofibroblastic character with sub-micrometer magnetoelastic vibrations.

The effect of sub-cellular mechanical loads on the behavior of fibroblasts was investigated using magnetoelastic (ME) materials, a type of material that produces mechanical vibrations when exposed to an external magnetic AC field. The integration of this functionality into implant surfaces could mitigate excessive fibrotic responses to many biomedical devices. By changing the profiles of the AC magnetic field, the amplitude, duration, and period of the applied vibrations was altered to understand the effect of each parameter on cell behavior. Results indicate fibroblast adhesion depends on the magnitude and total number of applied vibrations, and reductions in proliferative activity, cell spreading, and the expression of myofibroblastic markers occur in response to the vibrations induced by the ME materials. These findings suggest that the subcellular amplitude mechanical loads produced by ME materials could potentially remotely modulate myofibroblastic activity and limit undesirable fibrotic development.

Application of Composite Hydrogels to Control Physical Properties in Tissue Engineering and Regenerative Medicine.

The development of biomaterials for the restoration of the normal tissue structure⁻function relationship in pathological conditions as well as acute and chronic injury is an area of intense investigation. More recently, the use of tailored or composite hydrogels for tissue engineering and regenerative medicine has sought to bridge the gap between natural tissues and applied biomaterials more clearly. By applying traditional concepts in engineering composites, these hydrogels represent hierarchical structured materials that translate more closely the key guiding principles required for improved recovery of tissue architecture and functional behavior, including physical, mass transport, and biological properties. For tissue-engineering scaffolds in general, and more specifically in composite hydrogel materials, each of these properties provide unique qualities that are essential for proper augmentation and repair following disease and injury. The broad focus of this review is on physical properties in particular, static and dynamic mechanical properties provided by composite hydrogel materials and their link to native tissue architecture and, ultimately, tissue-specific applications for composite hydrogels.

Effect of incorporating clustered silica nanoparticles on the performance and biocompatibility of catechol-containing PEG-based bioadhesive.

A composite adhesive capable of inducing cellular infiltration was prepared by incorporating control clustered silica microparticle (MP) derived from the aggregation of silica nanoparticle (NP) into a catechol-terminated poly(ethylene glycol) bioadhesive (PEG-DA). Incorporation of MP into PEG-DA significantly improved the mechanical and adhesive properties of the bioadhesive. There was no statistical difference between the measured values for NP- and MP-incorporated adhesives, indicating that MP was equally as effective in enhancing the material properties of PEG-DA as NP. Most importantly, MP was significantly less cytotoxic when compared to NP when these particles were directly exposed to L929 fibroblast. When the adhesives were implanted subcutaneously in rats, MP-containing PEG-DA also exhibited reduced inflammatory responses, attracted elevated levels of regenerative M2 macrophage to its interface, and promoted cellular infiltration due to increased porosity within the adhesive network. Control clustered silica MP can be used to improve the performance and biocompatibility of PEG-based adhesive while minimizing undesirable cytotoxicity of silica NP.

Bioactive vapor deposited calcium-phosphate silica sol-gel particles for directing osteoblast behavior.

Silica-based materials are being developed and used for a variety of applications in orthopedic tissue engineering. In this work, we characterize the ability of a novel silica sol vapor deposition system to quickly modify biomaterial substrates and modulate surface hydrophobicity, surface topography, and composition. We were able to show that surface hydrophobicity, surface roughness, and composition could be rapidly modified. The compositional modification was directed towards generating apatitic-like surface mineral compositions (Ca/P ratios ∼1.30). Modified substrates were also capable of altering cell proliferation and differentiation behavior of preosteoblasts (MC3T3) and showed potential once optimized to provide a simple means to generate osteo-conductive substrates for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2135-2148, 2016.

Application of sub-micrometer vibrations to mitigate bacterial adhesion.

As a prominent concern regarding implantable devices, eliminating the threat of opportunistic bacterial infection represents a significant benefit to both patient health and device function. Current treatment options focus on chemical approaches to negate bacterial adhesion, however, these methods are in some ways limited. The scope of this study was to assess the efficacy of a novel means of modulating bacterial adhesion through the application of vibrations using magnetoelastic materials. Magnetoelastic materials possess unique magnetostrictive property that can convert a magnetic field stimulus into a mechanical deformation. In vitro experiments demonstrated that vibrational loads generated by the magnetoelastic materials significantly reduced the number of adherent bacteria on samples exposed to Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus suspensions. These experiments demonstrate that vibrational loads from magnetoelastic materials can be used as a post-deployment activated means to deter bacterial adhesion and device infection.

S-nitroso-N-acetylpenicillamine (SNAP) derivatization of peptide primary amines to create inducible nitric oxide donor biomaterials.

An S-nitroso-N-acetylpenicillamine (SNAP) derivatization approach was used to modify existing free primary amines found in fibrin (a natural protein-based biomaterial) to generate a controlled nitric oxide (NO) releasing scaffold material. The duration of the derivatization reaction affects the NO release kinetics, the induction of controlled NO-release, hydrophobicity, swelling behavior, elastic moduli, rheometric character, and degradation behavior. These properties were quantified to determine changes in fibrin hydrogels following covalent attachment of SNAP. NO-releasing materials exhibited minimal cytotoxicity when cultured with fibroblasts or osteoblasts. Cells maintained viability and proliferative character on derivatized materials as demonstrated by Live/Dead cell staining and counting. In addition, SNAP-derivatized hydrogels exhibited an antimicrobial character indicative of NO-releasing materials. SNAP derivatization of natural polymeric biomaterials containing free primary amines offers a means to generate inducible NO-releasing biomaterials for use as an antimicrobial and regenerative support for tissue engineering.

Development of vapor deposited silica sol-gel particles for use as a bioactive materials system.

Silica-based sol-gel and bioglass materials are used in a variety of biomedical applications including the surface modification of orthopedic implants and tissue engineering scaffolds. In this work, a simple system for vapor depositing silica sol-gel nano- and micro-particles onto substrates using nebulizer technology has been developed and characterized. Particle morphology, size distribution, and degradation can easily be controlled through key formulation and manufacturing parameters including water:alkoxide molar ratio, pH, deposition time, and substrate character. These particles can be used as a means to rapidly modify substrate surface properties, including surface hydrophobicity (contact angle changes >15°) and roughness (RMS roughness changes of up to 300 nm), creating unique surface topography. Ions (calcium and phosphate) were successfully incorporated into particles, and induced apatitie-like mineral formation upon exposure to simulated body fluid Preosteoblasts (MC3T3) cultured with these particles showed up to twice the adhesivity within 48 h when compared to controls, potentially indicating an increase in cell proliferation, with the effect likely due to both the modified substrate properties as well as the release of silica ions. This novel method has the potential to be used with implants and tissue engineering materials to influence cell behavior including attachment, proliferation, and differentiation via cell-material interactions to promote osteogenesis.

Fabrication of biocompatible, vibrational magnetoelastic materials for controlling cellular adhesion.

This paper describes the functionalization of magnetoelastic (ME) materials with Parylene-C coating to improve the surface reactivity to cellular response. Previous study has demonstrated that vibrating ME materials were capable of modulating cellular adhesion when activated by an externally applied AC magnetic field. However, since ME materials are not inherently biocompatible, surface modifications are needed for their implementation in biological settings. Here, the long-term stability of the ME material in an aqueous and biological environment is achieved by chemical-vapor deposition of a conformal Parylene-C layer, and further functionalized by methods of oxygen plasma etching and protein adsorption. In vitro cytotoxicity measurement and characterization of the vibrational behavior of the ME materials showed that Parylene-C coatings of 10 µm or greater could prevent hydrolytic degradation without sacrificing the vibrational behavior of the ME material. This work allows for long-term durability and functionality of ME materials in an aqueous and biological environment and makes the potential use of this technology in monitoring and modulating cellular behavior at the surface of implantable devices feasible.

Magnetoelastic materials as novel bioactive coatings for the control of cell adhesion.

Interfacial fibrosis is known to dramatically decrease the lifespan, stability, and function of biomedical implants and bone-anchored prosthetics. Bioactive coatings aimed at mitigating fibrous adhesions are one of the approaches to alleviate the problem. In this paper, we are developing a bioactive coating based upon a magnetoelastic (ME) material that vibrates in response to an ac magnetic field. In order to establish these coatings for this purpose, the ME material was first rendered bioactive through the sequential addition of polyurethane and chitosan thin films. Indirect live/dead assays were performed showing increased cell viability for polyurethane and chitosan-coated sensors compared to the uncoated controls. Direct adhesion experiments were performed to test the response of fibroblasts cultured on static and vibrated ME materials. Results showed cells adherent to static but not vibrated coatings. Detached cells showed no viability loss compared to controls. The finding that submicrometer ME vibrations can prevent cell adhesion in vitro without inducing cell death suggests the potential of these coatings to effectively control interfacial fibrosis. Future work will address the effect of vibrations on cell morphology and local gene expression in vitro, as well as fibrous tissue formation in vivo.

Real-time, in vivo investigation of mechanical stimulus on cells with remotely activated, vibrational magnetoelastic layers.

A system was developed for real-time, in vivo investigation of the relationship between local cell-level nano-mechanical perturbation and cell response to chemical-physical biomaterial surface properties. The system consisted of a magnetoelastic (ME) layer that could be remotely set to vibrate, at submicron levels, at a predetermined amplitude and profile. Experiments result indicated that submicron localized vibrations coupled with tailored biomaterial surface properties could selectively control cellular adhesion and possibly guide phenotypic gene expression. Practical application of this system includes modulation and monitoring of the surface of implantable biomaterials. The ME based vibrational system is the first of its kind for use in vitro for culture based mechanical testing, which could be readily deployed in situ as an in vivo system to apply local mechanical loads. It could be applied to specific implant surface sites and then subsequently sealed prior to long-term implantation. The potential advantage of this system over other similar approaches is that the system is translatable--the functional layer can serve as a "cellular workbench" material but could also be adapted and applied to the surface of implantable biomaterials and devices.

Role of carbonic anhydrase II in ectopic calcification.

INTRODUCTION: Osteopontin (OPN) is a potent inhibitor of ectopic calcification. Previous studies suggested that, in addition to blocking apatite crystal growth, OPN promoted regression of ectopic calcification by inducing the expression of acid-generating carbonic anhydrase II (CAR2) in monocyte-derived cells. METHODS: To test this hypothesis, OPN and CAR2 expression and calcification of subcutaneously implanted glutaraldehyde-fixed bovine pericardium (GFBP) were studied in CAR2 mutant mice. RESULTS: Consistent with previous studies in Black Swiss mice, GFBP calcified to a greater extent in OPN-deficient mice compared to wild types on the C57Bl/6 background. GFBP implanted in CAR2-deficient mice (CAR2(-/-)) were significantly more calcified than those implanted into wild-type mice (CAR2(+/+)) [37+/-5 vs. 20+/-6.5 microg Ca/mg tissue, respectively, at 30 days (P<.001), and 42+/-5 versus 20+/-4 microg Ca/mg tissue at 60 days, respectively (P<.001)]. On the other hand, OPN levels within and surrounding the implants were similar in CAR2(+/+) and CAR2(-/-) mice, suggesting that OPN expression in the absence of CAR2 was not sufficient to mitigate ectopic calcification. CONCLUSIONS: These results indicate that CAR2 expression is an important regulator of ectopic calcification, potentially by facilitating OPN mediated mineral regression.

Microporous nanofibrous fibrin-based scaffolds for bone tissue engineering.

The fibrotic response of the body to synthetic polymers limits their success in tissue engineering and other applications. Though porous polymers have demonstrated improved healing, difficulty in controlling their pore sizes and pore interconnections has clouded the understanding of this phenomenon. In this study, a novel method to fabricate natural polymer/calcium phosphate composite scaffolds with tightly controllable pore size, pore interconnection, and calcium phosphate deposition was developed. Microporous, nanofibrous fibrin scaffolds were fabricated using sphere-templating methods. Composite scaffolds were created by solution deposition of calcium phosphate on fibrin surfaces or by direct incorporation of nanocrystalline hydroxyapatite (nHA). The SEM results showed that fibrin scaffolds exhibited a highly porous and interconnected structure. Osteoblast-like cells, obtained from murine calvaria, attached, spread and showed a polygonal morphology on the surface of the biomaterial. Multiple cell layers and fibrillar matrix deposition were observed. Moreover, cells seeded on mineralized fibrin scaffolds exhibited significantly higher alkaline phosphatase activity as well as osteoblast marker gene expression compared to fibrin scaffolds and nHA incorporated fibrin scaffolds (0.25 and 0.5g). All types of scaffolds were degraded both in vitro and in vivo. Furthermore, these scaffolds promoted bone formation in a mouse calvarial defect model and the bone formation was enhanced by addition of rhBMP-2.

The influence of surface mineral and osteopontin on the formation and function of murine bone marrow-derived osteoclasts.

The phosphorylated glycoprotein osteopontin (OPN) is involved in the regulation of biomineralization under normal and pathological conditions. Its actions include inhibiting apatite crystal growth and promoting the formation and function of mineral resorbing cells, including osteoclasts (OCL). The purpose of this study was to develop stable apatitic mineral surfaces and determine their influence on OCL formation and mineral resorption from bone marrow macrophages derived from OPN wild-type (OPN+/+) and OPN deficient (OPN-/-) mice. We demonstrated that these mineral coatings were stable and supported bone marrow-derived macrophage differentiation to OCL under our culture conditions. Macrophages harvested from OPN-/- mice had a greater capacity to form OCL than macrophages from OPN+/+ mice when allowed to differentiate on tissue culture plastic. In contrast, when allowed to differentiate on a mineral surface, no difference in OCL formation was observed. Interestingly, OPN+/+ OCL were more efficient at mineral dissolution than OPN-/- OCL, and this difference was observed regardless of differentiating surface. Our results suggest that mineralized substrates as well as ability to synthesize OPN both control OCL function in our model system. The exact nature of these effects may be dependent on variables related to mineral substrate presentation.