Hyaluronic acid-British anti-Lewisite as a safer chelation therapy for the treatment of arthroplasty-related metallosis.

Cobalt-containing alloys are useful for orthopedic applications due to their low volumetric wear rates, corrosion resistance, high mechanical strength, hardness, and fatigue resistance. Unfortunately, these prosthetics release significant levels of cobalt ions, which was only discovered after their widespread implantation into patients requiring hip replacements. These cobalt ions can result in local toxic effects-including peri-implant toxicity, aseptic loosening, and pseudotumor-as well as systemic toxic effects-including neurological, cardiovascular, and endocrine disorders. Failing metal-on-metal (MoM) implants usually necessitate painful, risky, and costly revision surgeries. To treat metallosis arising from failing MoM implants, a synovial fluid-mimicking chelator was designed to remove these metal ions. Hyaluronic acid (HA), the major chemical component of synovial fluid, was functionalized with British anti-Lewisite (BAL) to create a chelator (BAL-HA). BAL-HA effectively binds cobalt and rescues in vitro cell vitality (up to 370% of cells exposed to IC50 levels of cobalt) and enhances the rate of clearance of cobalt in vivo (t1/2 from 48 h to 6 h). A metallosis model was also created to investigate our therapy. Results demonstrate that BAL-HA chelator system is biocompatible and capable of capturing significant amounts of cobalt ions from the hip joint within 30 min, with no risk of kidney failure. This chelation therapy has the potential to mitigate cobalt toxicity from failing MoM implants through noninvasive injections into the joint.

Phosphate graphene as an intrinsically osteoinductive scaffold for stem cell-driven bone regeneration.

Synthetic, resorbable scaffolds for bone regeneration have potential to transform the clinical standard of care. Here, we demonstrate that functional graphenic materials (FGMs) could serve as an osteoinductive scaffold: recruiting native cells to the site of injury and promoting differentiation into bone cells. By invoking a Lewis acid-catalyzed Arbuzov reaction, we are able to functionalize graphene oxide (GO) to produce phosphate graphenes (PGs) with unprecedented control of functional group density, mechanical properties, and counterion identity. In aqueous environments, PGs release inducerons, including Ca2+ and PO43- Calcium phosphate graphene (CaPG) intrinsically induces osteogenesis in vitro and in the presence of bone marrow stromal cells (BMSCs), can induce ectopic bone formation in vivo. Additionally, an FGM can be made by noncovalently loading GO with the growth factor recombinant human bone morphogenetic protein 2 (rhBMP-2), producing a scaffold that induces ectopic bone formation with or without BMSCs. The FGMs reported here are intrinsically inductive scaffolds with significant potential to revolutionize the regeneration of bone.

One-Shot Synthesis of Peptide Amphiphiles with Applications in Directed Graphenic Assembly.

High molecular weight, synthetic block copolypeptides that self-assemble are in high demand for biomedical applications. The current standard method for synthesis of block copolypeptides is the controlled ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydride (NCA) monomers, where block architectures can be created by sequential NCA monomer addition. Recently, researchers have focused on developing reaction conditions and initiation systems that make NCA ROP more convenient, particularly for interdisciplinary labs without designated polypeptide facilities. In an effort to further simplify and increase the convenience of polypeptide synthesis, we developed a one-shot copolymerization strategy that allows access to block copolypeptides by capitalizing on the inherently faster reactivity of NCA monomers, compared to NTA (N-thiocarboxyanhydride) monomers. For the first time, we combine an NCA and NTA monomer in one reaction to kinetically promote block copolypeptide formation, providing a convenient alternative to sequential monomer addition. The controlled nature of this copolymerization technique is supported by a molecular weight that is modulated by the concentration of the initiator and low dispersities. We used this one-shot copolymerization to synthesize p(lysine)-b-p(leucine), a known peptide amphiphile (PA). Our one-shot PAs are antimicrobial and can spontaneously form ordered, micron-scale assemblies. Covalent conjugation of one-shot PAs to a graphenic backbone results in a functional graphenic material (FGM) with a self-assembled morphology, paving the way for creation of sophisticated FGM scaffolds with polypeptide-templated, hierarchical order. Overall, we demonstrate that this novel, one-shot copolymerization strategy produces functional copolypeptides with macroscopic sequence control.

Calcium phosphate graphene and Ti3C2Tx MXene scaffolds with osteogenic and antibacterial properties.

Bioactive degradable scaffolds that facilitate bone healing while fighting off initial bacterial infection have the potential to change established strategies of dealing with traumatic bone injuries. To achieve this a composite material made from calcium phosphate graphene (CaPG), and MXene was synthesized. CaPG was created by functionalizing graphene oxide with phosphate groups in the presence of CaBr with a Lewis acid catalyst. Through this transformation, Ca2+ and PO4 3- inducerons are released as the material degrades thereby aiding in the process of osteogenesis. The 2D MXene sheets, which have shown to have antibacterial properties, were made by etching the Al from a layered Ti3AlC2 (MAX phase) using HF. The hot-pressed scaffolds made of these materials were designed to combat the possibility of infection during initial surgery and failure of osteogenesis to occur. These two failure modes account for a large percentage of issues that can arise during the treatment of traumatic bone injuries. These scaffolds were able to retain induceron-eluting properties in various weight percentages and bring about osteogenesis with CaPG alone and 2 wt% MXene scaffolds demonstrating increased osteogenic activity as compared to no treatment. Additionally, added MXene provided antibacterial properties that could be seen at as little as 2 wt%. This CaPG and MXene composite provides a possible avenue for developing osteogenic, antibacterial materials for treating bone injuries.

Environmental and health impacts of functional graphenic materials and their ultrasonically altered products.

Graphenic materials have excited the scientific community due to their exciting mechanical, thermal, and optoelectronic properties for a potential range of applications. Graphene and graphene derivatives have demonstrated application in areas stretching from composites to medicine; however, the environmental and health impacts of these materials have not been sufficiently characterized. Graphene oxide (GO) is one of the most widely used graphenic derivatives due to a relatively easy and scalable synthesis, and the ability to tailor the oxygen containing functional groups through further chemical modification. In this paper, ecological and health impacts of fresh and ultrasonically altered functional graphenic materials (FGMs) were investigated. Model organisms, specifically Escherichia coli, Bacillus subtilis, and Caenorhabditis elegans, were used to assess the consequences of environmental exposure to fresh and ultrasonically altered FGMs. FGMs were selected to evaluate the environmental effects of aggregation state, degree of oxidation, charge, and ultrasonication. The major findings indicate that bacterial cell viability, nematode fertility, and nematode movement were largely unaffected, suggesting that a wide variety of FGMs may not pose significant health and environmental risks.

Ultra-low binder content 3D printed calcium phosphate graphene scaffolds as resorbable, osteoinductive matrices that support bone formation in vivo.

Bone regenerative engineering could replace autografts; however, no synthetic material fulfills all design criteria. Nanocarbons incorporated into three-dimensional printed (3DP) matrices can improve properties, but incorporation is constrained to low wt%. Further, unmodified nanocarbons have limited osteogenic potential. Functionalization to calcium phosphate graphene (CaPG) imparts osteoinductivity and osteoconductivity, but loading into matrices remained limited. This work presents ultra-high content (90%), 3DP-CaPG matrices. 3DP-CaPG matrices are highly porous (95%), moderately stiff (3 MPa), and mechanically robust. In vitro, they are cytocompatible and induce osteogenic differentiation of human mesenchymal stem cells (hMSCs), indicated by alkaline phosphatase, mineralization, and COL1α1 expression. In vivo, bone regeneration was studied using a transgenic fluorescent-reporter mouse non-union calvarial defect model. 3DP-CaPG stimulates cellular ingrowth, retains donor cells, and induces osteogenic differentiation. Histology shows TRAP staining around struts, suggesting potential osteoclast activity. Apparent resorption of 3DP-CaPG was observed and presented no toxicity. 3DP-CaPG represents an advancement towards a synthetic bone regeneration matrix.

Hyaluronic Acid-Based Antibacterial Hydrogels for Use as Wound Dressings.

Wound dressings have been shifting toward a more active role in the wound-healing process. Hydrated environments with additives to aid in the healing process are currently being explored through the application of hydrocolloid dressings. However, these moist healing environments are also ideal for bacterial growth, leading to the widespread use of antibiotics with concerns of antibiotic resistance and toxicity. To overcome this concern, we present a hydrogel wound dressing consisting of hyaluronic acid (HA) cross-linked with gentamicin. This hydrogel treats bacterial infection locally, lowering the effective dose and reducing the concerns of antibiotic resistance and systemic exposure. Changing the cross-linking density, by using varied amounts of a cross-linker, created gels that provided a sustained release of gentamicin for up to 9 days with a range of adhesive and cohesive properties. Overall, this HA hydrogel could provide an important solution in treating local infection in burns and other dermal injuries.

The Blanket Effect: How Turning the World Upside Down Reveals the Nature of Graphene Oxide Cytocompatibility.

Extensive cytocompatibility testing of 2D nanocarbon materials including graphene oxide (GO) has been performed, but results remain contradictory. Literature has yet to account for settling-although sedimentation is visible to the eye and physics suggests that even individual graphenic flakes will settle. To investigate settling, a series of functional graphenic materials (FGMs) with differing oxidation levels, functionalities, and physical dimensions are synthesized. Though zeta potential indicates colloidal stability, significant gravitational settling of the FGMs is theoretically and experimentally demonstrated. By creating a setup to culture cells in traditional and inverted orientations in the same well, a "blanket effect" is demonstrated in which FGMs settle out of solution and cover cells at the bottom of the well, ultimately reducing viability. Inverted cells protected from the blanket effect are unaffected. Therefore, these results demonstrate that settling is a crucial factor that must be considered for FGM cytocompatibility experiments.

Tunable, bacterio-instructive scaffolds made from functional graphenic materials.

The balance of bacterial populations in the human body is critical for human health. Researchers have aimed to control bacterial populations using antibiotic substrates. However, antibiotic materials that non-selectively kill bacteria can compromise health by eliminating beneficial bacteria, which leaves the body vulnerable to colonization by harmful pathogens. Due to their chemical tunablity and unique surface properties, graphene oxide (GO)-based materials - termed "functional graphenic materials" (FGMs) - have been previously designed to be antibacterial but have the capacity to actively adhere and instruct probiotics to maintain human health. Numerous studies have demonstrated that negatively and positively charged surfaces influence bacterial adhesion through electrostatic interactions with the negatively charged bacterial surface. We found that tuning the surface charge of FGMs provides an avenue to control bacterial attachment without compromising vitality. Using E. coli as a model organism for Gram-negative bacteria, we demonstrate that negatively charged Claisen graphene (CG), a reduced and carboxylated FGM, is bacterio-repellent through electrostatic repulsion with the bacterial surface. Though positively charged poly-l-lysine (PLL) is antibacterial when free in solution by inserting into the bacterial cell wall, here, we found that covalent conjugation of PLL to CG (giving PLLn-G) masks the antimicrobial activity of PLL by restricting polypeptide mobility. This allows the immobilized positive charge of the PLLn-Gs to be leveraged for E. coli adhesion through electrostatic attraction. We identified the magnitude of positive charge of the PLLn-G conjugates, which is modulated by the length of the PLL peptide, as an important parameter to tune the balance between the opposing forces of bacterial adhesion and proliferation. We also tested adhesion of Gram-positive B. subtilis to these FGMs and found that the effect of FGM charge is less pronounced. B. subtilis adheres nondiscriminatory to all FGMs, regardless of charge, but adhesion is scarce and localized. Overall, this work demonstrates that FGMs can be tuned to selectively control bacterial response, paving the way for future development of FGM-based biomaterials as bacterio-instructive scaffolds through careful design of FGM surface chemistry.

Graphene-Based Biomaterials for Bone Regenerative Engineering: A Comprehensive Review of the Field and Considerations Regarding Biocompatibility and Biodegradation.

Graphene and its derivatives have continued to garner worldwide interest due to their unique characteristics. Having expanded into biomedical applications, there have been efforts to employ their exceptional properties for the regeneration of different tissues, particularly bone. This article presents a comprehensive review on the usage of graphene-based materials for bone regenerative engineering. The graphene family of materials (GFMs) are used either alone or in combination with other biomaterials in the form of fillers in composites, coatings for both scaffolds and implants, or vehicles for the delivery of various signaling and therapeutic agents. The applications of the GFMs in each of these diverse areas are discussed and emphasis is placed on the characteristics of the GFMs that have implications in this regard. In tandem and of importance, this article evaluates the safety and biocompatibility of the GFMs and carefully elucidates how various factors influence the biocompatibility and biodegradability of this new class of nanomaterials. In conclusion, the challenges and opportunities regarding the use of the GFMs in regenerative engineering applications are discussed, and future perspectives for the developments in this field are proposed.

Bioactive, Ion-Releasing PMMA Bone Cement Filled with Functional Graphenic Materials.

Graphene oxide and functionalized graphenic materials (FGMs) have promise as platforms for imparting programmable bioactivity to poly(methyl methacrylate) (PMMA)-based bone cement. To date, however, graphenic fillers have only been feasible in PMMA cements at extremely low loadings, limiting the bioactive effects. At higher loadings, graphenic fillers decrease cement strength by aggregating and interfering with curing process. Here, these challenges are addressed by combining bioactive FGM fillers with a custom cement formulation. These cements contain an order of magnitude more graphenic filler than previous reports. Even at 1 wt% FGM, these cements have compressive strengths of 78- 88 MPa, flexural strengths of 74-81 MPa, and flexural stiffnesses of 1.8-1.9 GPa, surpassing the ASTM requirements for bone cement and competing with traditional PMMA cement. Further, by utilizing designer FGMs with programmed bioactivity, these cements demonstrate controlled release of osteogenic calcium ions (releasing a total of 5 ± 2 µmol of Ca2+ per gram of cement over 28 d) and stimulate a 290% increase in expression of alkaline phosphatase in human mesenchymal stem cells in vitro. Also, design criteria are described to guide creation of future generations of bone cements that utilize FGMs as platforms to achieve dynamic biological activity.

Polyester functional graphenic materials as a mechanically enhanced scaffold for tissue regeneration.

Traditional metal implants such as titanium, cobalt, and chromium have found wide utility in medicine; however, these come with a risk of toxicity. To overcome metal-related toxicity and enable degradability, polyesters including polycaprolactone (PCL), polylactic acid (PLA), and polyglycolic acid (PGA) show promise for the replacement of various biomedical applications of metals due to their accepted biocompatibility and FDA approval. However, polyesters are less stiff than their metallic counterparts, limiting their application to non-load bearing injury sites, such as fixation hardware for fingers. To improve mechanical properties, graphene oxide (GO)-polyester composites are a promising class of biodegradable scaffolds. Initial reports of these composites are encouraging, but mechanical properties still fall short. Traditional composites rely on non-covalent association between GO and the polyesters, which often leads to failure at the interface and weakens the overall strength of the material. Herein, we present a strategy for attachment of these FDA-approved polyesters onto a derivative of GO using a robust covalent bond. By covalently functionalizing the graphenic backbone with polyesters and without metal catalysts, we create functional graphenic materials (FGMs) to not only simultaneously retain biodegradability and compatibility, but also mechanically strengthen PCL, PLA, and PGA; we observed an average increase in the Young's modulus of over 140% compared to the graphenic backbone. These polyester-functionalized FGMs are a promising platform technology for tissue implants.

Acid Mine Drainage Remediation: Aluminum Chelation Using Functional Graphenic Materials.

Acid mine drainage (AMD) is a pervasive source of metal pollution that severely impacts freshwater ecosystems and has a direct impact on human health. Conventional active and passive methods work very well for removing iron in AMD remediation, which is typically the highest metallic impurity. However, conventional passive remediation fails to remove all aluminum, which has severe ecological implications. Removal of aluminum ions using chelation, which traditionally uses small molecules that bind metals tightly for sequestration, holds promise. Yet, chelation strategies are limited because once introduced into surface water, small molecules are difficult to reclaim and often persist in the environment as pollutants. To address this, we have designed six unique scaffolds based on functional graphenic materials (FGMs) to create nonsoluble materials that could be placed at the end of a passive remediation process to remove persistent aluminum. When tested for efficacy, all six FGMs successfully demonstrated a reversible capacity to remove aluminum from acidic water, chelating up to 21 µg of Al/mg of FGM. Furthermore, when they were exposed to E. coli as an approximation for environmental compatibility, viability was unaffected, even at high concentrations, suggesting these FGMs are nontoxic and viable candidates for passive chelation-based remediation.

Modular functionalization of carbon nanotubes and fullerenes.

A series of highly efficient, modular zwitterion-mediated transformations have been developed which enable diverse functionalization of carbon nanotubes (CNTs, both single-walled and multi-walled) and fullerenes. Three functionalization strategies are demonstrated. (1) Trapping the charged zwitterion intermediate with added nucleophiles allows a variety of functional groups to be installed on the fullerenes and carbon nanotubes in a one-pot reaction. (2) Varying the electrophile from dimethyl acetylenedicarboxylate to other disubstituted esters provides CNTs functionalized with chloroethyl, allyl, and propargyl groups, which can further undergo S(N)2 substitution, thiol addition, or 1,3-dipolar cycloaddition reactions. (3) Postfunctionalization transformations on the cyclopentenones (e.g., demethylation and saponification) of the CNTs lead to demethylated or hydrolyzed products, with high solubility in water (1.2 mg/mL for MWCNTs). CNT aqueous dispersions of the latter derivatives are stable for months and have been successfully utilized in preparation of CNT-poly(ethylene oxide) nanocomposite via electrospinning. Large-scale MWCNT (10 g) functionalization has also been demonstrated to show the scalability of the zwitterion reaction. In total we present a detailed account of diverse CNT functionalization under mild conditions (60 degrees C, no strong acids/bases, or high pressure) and with high efficiency (1 functional group per 10 carbon atoms for SWCNTs), which expand the utility of these materials.

Covalent conjugation of bioactive peptides to graphene oxide for biomedical applications.

Graphene is a valuable material in biomedical implant applications due to its mechanical integrity, long-range order, and conductivity; but graphene must be chemically modified to increase biocompatibility and maximize functionality in the body. Here, we developed a foundational synthetic method for covalently functionalizing a reduced GO with bioactive molecules, focusing on synthetic peptides that have shown osteogenic or neurogenic capability as a prototypical example. X-ray photoelectron spectroscopy provides evidence that the peptide is covalently linked to the graphenic backbone. These peptide-graphene (Pep-G) conjugate materials can be processed into mechanically robust, three-dimensional constructs. Differences in their electrostatic charges allow the Pep-G conjugates to form self-assembled, layer-by-layer coatings. Further, the Pep-G conjugates are cytocompatible and electrically conductive, leading us to investigate their potential as regenerative scaffolds, as conductive surfaces can stimulate bone and nerve regeneration. Notably, PC12 cells grown on an electrically stimulated Pep-G scaffold demonstrated enhanced adhesion and neurite outgrowth compared to the control. The functionalization strategy developed here can be used to conjugate a wide variety of bioactive molecules to graphene oxide to create cell-instructive surfaces for biomedical scaffold materials.

Functional Graphenic Materials That Seal Condenser Tube Leaks in Situ.

Undesirable condenser tube leaks frequently occur in power plants, resulting in reduced power output, increased burden on downstream systems, and substantial revenue losses. Current techniques such as wood flour provide temporary in situ remediation but lack adhesive properties to form stable seals. Here, we report the development of in situ sealants for long-term defect repair. The carboxylic acids on graphene oxides and Claisen graphene were used as chemical handles to covalently install a bio-inspired, adhesive catechol, generating a class of functional graphenic material (FGM) sealants. FGM sealants outperformed unfunctionalized scaffolds with enhanced antimicrobial activity to prevent fouling (up to 55% reduction) and superior cohesive properties to promote stable seals. Further, FGM sealants were adhesive, effectively sealing defects in a model experiment, whereas unfunctionalized scaffolds did not display any sealant capacity.

Injectable amine functionalized graphene and chondroitin sulfate hydrogel with potential for cartilage regeneration.

Damaged cartilage does not readily heal and often requires surgical intervention that only modestly improves outcomes. A synthetic material that could be injected and covalently crosslinked in situ to form a bioactive, mechanically robust scaffold that promotes stem cell chondrogenic differentiation holds promise for next-generation treatment of cartilage lesions. Here, Johnson-Claisen rearrangement chemistry was performed on graphene oxide (GO) to enable functionalization with a primary amine covalently bound to the graphenic backbone through a chemically stable linker. The primary amines are used to form covalent crosslinks with chondroitin sulfate, an important component of cartilage that promotes regeneration, to form a hydrogel (EDAG-CS). The EDAG-CS system gels in situ within 10 min, and the graphenic component imparts improved mechanical properties, including stiffness (320% increase) and toughness (70% increase). EDAG-CS hydrogels are highly porous, resistant to degradation, and enable the growth of human mesenchymal stem cells and their deposition of collagen matrix. This system has potential to improve clinical outcomes of patients with cartilage damage.

Covalently-controlled drug delivery via therapeutic methacrylic tissue adhesives.

Medical cyanoacrylate adhesives have the potential to eliminate the need for sutures but face challenges to widespread implementation due to their brittleness and release of formaldehyde upon degradation. To overcome these limitations, we used molecular design to create therapeutic methacrylic (TMA) monomers to impart tunable mechanical properties, decreased formaldehyde release, and covalently-controlled bioactivity to commercial cyanoacrylate adhesives. The small molecule therapeutics ibuprofen, acetaminophen, and benzocaine were covalently tethered to the carbonyl of methacrylate using anhydride, ester, and amide bonds. When these TMAs were incorporated into n-butyl cyanoacrylate (BCA) tissue adhesives, the resulting TMA-BCA materials provided release of the therapeutics across a range of time scales according to the reactivity of the tether bond to hydrolysis. The anhydride-tether TMA-BCA adhesive delivered ibuprofen on the same order of magnitude and time scale as topical medications (12 ± 6 mg per g adhesive after 3.4 h). TMA-BCA adhesives also produced less formaldehyde than standard BCA adhesive, showed promising cytocompatibility, and adhered effectively to porcine skin. Further, the anhydride, ester, and amide tether TMA-BCA adhesives exhibited a range of shear moduli, with those containing rigid aromatic amide groups being stiffer, and those with flexible alkyl segments being less stiff, which could enable these adhesives to be tailored to match the mechanical properties of target tissues. The amide-tether TMA-BCA adhesive also showed a 219% increase in toughness compared to BCA. Overall, TMAs represent a platform technology that can be used to build adaptable and bioactive tissue adhesives.

Graphene oxide as a scaffold for bone regeneration.

Graphene oxide (GO), the oxidized form of graphene, holds great potential as a component of biomedical devices, deriving utility from its ability to support a broad range of chemical functionalities and its exceptional mechanical, electronic, and thermal properties. GO composites can be tuned chemically to be biomimetic, and mechanically to be stiff yet strong. These unique properties make GO-based materials promising candidates as a scaffold for bone regeneration. However, questions still exist as to the compatibility and long-term toxicity of nanocarbon materials. Unlike other nanocarbons, GO is meta-stable, water dispersible, and autodegrades in water on the timescale of months to humic acid-like materials, the degradation products of all organic matter. Thus, GO offers better prospects for biological compatibility over other nanocarbons. Recently, many publications have demonstrated enhanced osteogenic performance of GO-containing composites. Ongoing work toward surface modification or coating strategies could be useful to minimize the inflammatory response and improve compatibility of GO as a component of medical devices. Furthermore, biomimetic modifications could offer mechanical and chemical environments that encourage osteogenesis. So long as care is given to assure their safety, GO-based materials may be poised to become the next generation scaffold for bone regeneration. WIREs Nanomed Nanobiotechnol 2017, 9:e1437. doi: 10.1002/wnan.1437 For further resources related to this article, please visit the WIREs website.

In It for the Long Haul: The Cytocompatibility of Aged Graphene Oxide and Its Degradation Products.

Synthetic biomaterials are poised to transform medicine; however, current synthetic options have yet to ideally recapitulate the desirable properties of native tissue. Thus, the development of new synthetic biomaterials remains an active challenge. Due to its excellent properties, including electrical conductivity, water dispersibility, and capacity for functionalization, graphene oxide (GO) holds potential for myriads of applications, including biological devices. While many studies have evaluated the compatibility of freshly prepared GO, understanding the compatibility of GO as it ages in an aqueous environment is crucial for its safe implementation in long-term biological applications. This is a critical disconnect, as GO has been shown to undergo an autodegradation pathway in aqueous conditions, dynamically changing its composition and structure while producing degradation products. Thus, the long-term cytocompatibility of GO is investigated by "aging" GO over time in water and accelerating aging and decomposition via sonication. While age affects the composition and size of GO, it has no effect on cellular vitality and does not alter subcellular structures or DNA melting. Overall, GO is cytocompatible throughout the process of aging, beginning to demonstrate that GO may be utilized for long-term in vivo applications such as implanted tissue engineered scaffolds or biosensors.