Recent trends in 3D bioprinting technology for skeletal muscle regeneration.

Skeletal muscle is a pro-regenerative tissue, that utilizes a tissue-resident stem cell system to effect repair upon injury. Despite the demonstrated efficiency of this system in restoring muscle mass after many acute injuries, in conditions of severe trauma such as those evident in volumetric muscle loss (VML) (>20 % by mass), this self-repair capability is unable to restore tissue architecture, requiring interventions which currently are largely surgical. As a possible alternative, the generation of artificial muscle using tissue engineering approaches may also be of importance in the treatment of VML and muscle diseases such as dystrophies. Three-dimensional (3D) bioprinting has been identified as a promising technique for regeneration of the complex architecture of skeletal muscle. This review discusses existing treatment strategies following muscle damage, recent progress in bioprinting techniques, the bioinks used for muscle regeneration, the immunogenicity of scaffold materials, and in vitro and in vivo maturation techniques for 3D bio-printed muscle constructs. The pros and cons of these bioink formulations are also highlighted. Finally, we present the current limitations and challenges in the field and critical factors to consider for bioprinting approaches to become more translationa and to produce clinically relevant engineered muscle. STATEMENT OF SIGNIFICANCE: This review discusses the physiopathology of muscle injuries and existing clinical treatment strategies for muscle damage, the types of bioprinting techniques that have been applied to bioprinting of muscle, and the bioinks commonly used for muscle regeneration. The pros and cons of these bioinks are highlighted. We present a discussion of existing gaps in the literature and critical factors to consider for the translation of bioprinting approaches and to produce clinically relevant engineered muscle. Finally, we provide insights into what we believe will be the next steps required before the realization of the application of tissue-engineered muscle in humans. We believe this manuscript is an insightful, timely, and instructive review that will guide future muscle bioprinting research from a fundamental construct creation approach, down a translational pathway to achieve the desired impact in the clinic.

Versatile xanthan gum-based support bath material compatible with multiple crosslinking mechanisms: rheological properties, printability, and cytocompatibility study.

Extrusion-based bioprinting is a promising technology for the fabrication of complex three-dimensional (3D) tissue-engineered constructs. To further improve the printing accuracy and provide mechanical support during the printing process, hydrogel-based support bath materials have been developed. However, the gel structure of some support bath materials can be compromised when exposed to certain bioink crosslinking cues, hence their compatibility with bioinks can be limited. In this study, a xanthan gum-based composite support material compatible with multiple crosslinking mechanisms is developed. Different support bath materials can have different underlying polymeric structures, for example, particulate suspensions and polymer solution with varying supramolecular structure) and these properties are governed by a variety of different intermolecular interactions. However, common rheological behavior can be expected because they have similar demonstrated performance and functionality. To provide a detailed exploration/identification of the common rheological properties expressed by different support bath materials from a unified perspective, benchmark support bath materials from previous studies were prepared. A comparative rheological study revealed both the structural and shear behavior characteristics shared by support bath materials, including yield stress, gel complex moduli, shear-thinning behavior, and self-healing properties. Gel structural stability and functionality of support materials were tested in the presence of various crosslinking stimuli, confirming the versatility of the xanthan-based support material. We further investigated the effect of support materials and the diameter of extrusion needles on the printability of bioinks to demonstrate the improvement in bioink printability and structural integrity. Cytotoxicity and cell encapsulation viability tests were carried out to confirm the cell compatibility of the xanthan gum-based support bath material. We propose and demonstrate the versatility and compatibility of the novel support bath material and provide detailed new insight into the essential properties and behavior of these materials that serve as a guide for further development of support bath-based 3D bioprinting.

Organosilica Nanosensors for Monitoring Spatiotemporal Changes in Oxygen Levels in Bacterial Cultures.

Oxygen plays a central role in aerobic metabolism, and while many approaches have been developed to measure oxygen concentration in biological environments over time, monitoring spatiotemporal changes in dissolved oxygen levels remains challenging. To address this, we developed a ratiometric core-shell organosilica nanosensor for continuous, real-time optical monitoring of oxygen levels in biological environments. The nanosensors demonstrate good steady state characteristics (KpSV = 0.40 L/mg, R2 = 0.95) and respond reversibly to changes in oxygen concentration in buffered solutions and report similar oxygen level changes in response to bacterial cell growth (Escherichia coli) in comparison to a commercial bulk optode-based sensing film. We further demonstrated that the oxygen nanosensors could be distributed within a growing culture of E. coli and used to record oxygen levels over time and in different locations within a static culture, opening the possibility of spatiotemporal monitoring in complex biological systems.

3D Functional Neuronal Networks in Free-Standing Bioprinted Hydrogel Constructs.

The composition, elasticity, and organization of the extracellular matrix within the central nervous system contribute to the architecture and function of the brain. From an in vitro modeling perspective, soft biomaterials are needed to mimic the 3D neural microenvironments. While many studies have investigated 3D culture and neural network formation in bulk hydrogel systems, these approaches have limited ability to position cells to mimic sophisticated brain architectures. In this study, cortical neurons and astrocytes acutely isolated from the brains of rats are bioprinted in a hydrogel to form 3D neuronal constructs. Successful bioprinting of cellular and acellular strands in a multi-bioink approach allows the subsequent formation of gray- and white-matter tracts reminiscent of cortical structures. Immunohistochemistry shows the formation of dense, 3D axon networks. Calcium signaling and extracellular electrophysiology in these 3D neuronal networks confirm spontaneous activity in addition to evoked activities under pharmacological and electrical stimulation. The system and bioprinting approaches are capable of fabricating soft, free-standing neuronal structures of different bioink and cell types with high resolution and throughput, which provide a promising platform for understanding fundamental questions of neural networks, engineering neuromorphic circuits, and for in vitro drug screening.

Extrusion 3D bioprinting of functional self-supporting neural constructs using a photoclickable gelatin bioink.

Manyin vitromodels of neural physiology utilize neuronal networks established on two-dimensional substrates. Despite the simplicity of these 2D neuronal networks, substrate stiffness may influence cell morphology, network interactions and how neurons communicate and function. With this perspective, three-dimensional (3D) gel encapsulation is a powerful to recapitulating aspects ofin vivofeatures, yet such an approach is often limited in terms of the level of resolution and feature size relevant for modelling aspects of brain architecture. Here, we report 3D bioplotting of rat primary cortical neural cells using a hydrogel system comprising gelatin norbornene (GelNB) and poly (ethylene glycol) dithiol (PEGdiSH). This bioink benefits from a rapid photo-click chemistry, yielding eight-layer crosshatch neural scaffolds and a filament width of 350µm. The printability of this system depends on hydrogel concentration, printing temperature, extrusion pressure and speed. These parameters were studied via quantitative comparison between rheology and filament dimensions to determine the optimal printing conditions. Under optimal conditions, cell viability of bioprinted primary cortical neurons at day 1 (68 ± 2%) and at day 7 (68 ± 1%) were comparable to the 2D control group (72 ± 7%). The present study relates material rheology and filament dimensions to generate compliant free-standing neural constructs through bioplotting of low-concentration GelNB-PEGdiSH, which may provide a step forward to study 3D neuronal function and network formation.

Review: Nanomaterials for Reactive Oxygen Species Detection and Monitoring in Biological Environments.

Reactive oxygen species (ROS) and dissolved oxygen play key roles across many biological processes, and fluorescent stains and dyes are the primary tools used to quantify these species in vitro. However, spatio-temporal monitoring of ROS and dissolved oxygen in biological systems are challenging due to issues including poor photostability, lack of reversibility, and rapid off-site diffusion. In particular, ROS monitoring is hindered by the short lifetime of ROS molecules and their low abundance. The combination of nanomaterials and fluorescent detection has led to new opportunities for development of imaging probes, sensors, and theranostic products, because the scaffolds lead to improved optical properties, tuneable interactions with cells and media, and ratiometric sensing robust to environmental drift. In this review, we aim to critically assess and highlight recent development in nanosensors and nanomaterials used for the detection of oxygen and ROS in biological systems, and their future potential use as diagnosis tools.

Poly(HPMA-co-NIPAM) copolymer as an alternative to polyethylene glycol-based pharmacokinetic modulation of therapeutic proteins.

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.

Stability and Performance Study of Fluorescent Organosilica pH Nanosensors.

Long-term stability and function are key challenges for optical nanosensors operating in complex biological environments. While much focus is rightly placed on issues related to specificity, sensitivity, reversibility, and response time, many nanosensors are not capable of transducing accurate results over prolonged time periods. Sensors could fail over time due to the degradation of scaffold material, degradation of signaling dyes and components, or a combination of both. It is critical to investigate how such degradative processes affect sensor output, as the consequences could be severe. Herein, we used fluorescent core-shell organosilica pH nanosensors as a model system, incubating them in a range of common aqueous solutions over time at different temperatures, and then searched for changes in fluorescence signal, particle size, and evidence of silica degradation. We found that these ratiometric nanosensors produced stable optical signals after aging for 30 days at 37 °C in standard saline buffers with and without 10% fetal bovine serum, and without any evidence of material degradation. Next, we evaluated their performance as real-time pH nanosensors in bacterial suspension cultures, observing a close agreement with a pH electrode for control nanosensors, yet observing obvious deviations in signal based on the aging conditions. The results show that while the organosilica scaffold does not degrade appreciably over time, careful selection of dyes and further systematic investigations into the effects of salt and protein levels are required to realize long-term stable nanosensors.

Modulation of substrate van der Waals forces using varying thicknesses of polymer overlayers.

Thin polymeric coatings are commonly used for altering surface properties and modulating the interfacial performance of materials. Possible contributions from the substrate to the interfacial forces and effects are, however, usually ignored and are not well understood, nor is it established how the coating thickness modulates and eventually eliminates contributions from substrates to the van der Waals (vdW) interfacial force. In this study we quantified, by colloid-probe atomic force microscope (AFM) and by theoretical calculations, the interfacial vdW contributions from substrates acting through ethanol plasma polymer (EtOHpp) coatings of a range of thicknesses on Au and Si bulk materials. In approach force curves against EtOHpp-coated Au substrates the magnitude of the vdW force decreased as the EtOHpp coating thickness increased to 18 nm and then plateaued with further increases in coating thickness, providing direct evidence for a contribution to the total interfacial vdW force from the Au substrate acting through thin coatings. The experimental observations accord with theoretical calculations of the thickness dependence of Hamaker coefficients derived from rigorous simulation using the Lifshitz theory. In addition, the measured forces agree well with theoretical predictions including correction for finite roughness. Thus, our experimental and theoretical results establish how the thickness of polymer thin film coatings modulates the total interfacial vdW force and how this can be used to tune the net vdW force so as to either contain a large substrate contribution or arise predominantly from the polymeric overlayer. Our findings enable rational design of coating thickness to tailor interfacial interactions and material performance.

Biological Considerations in Scaling Up Therapeutic Cell Manufacturing.

Cell therapeutics - using cells as living drugs - have made advances in many areas of medicine. One of the most clinically studied cell-based therapy products is mesenchymal stromal cells (MSCs), which have shown promising results in promoting tissue regeneration and modulating inflammation. However, MSC therapy requires large numbers of cells, the generation of which is not feasible via conventional planar tissue culture methods. Scale-up manufacturing methods (e.g., propagation on microcarriers in stirred-tank bioreactors), however, are not specifically tailored for MSC expansion. These processes may, in principle, alter the cell secretome, a vital component underlying the immunosuppressive properties and clinical effectiveness of MSCs. This review outlines our current understanding of MSC properties and immunomodulatory function, expansion in commercial manufacturing systems, and gaps in our knowledge that need to be addressed for effective up-scaling commercialization of MSC therapy.

Bacterial membrane permeability of antimicrobial polymethacrylates: Evidence for a complex mechanism from super-resolution fluorescence imaging.

Amphiphilic polymers bearing cationic moieties are an emerging alternative to traditional antibiotics given their broad-spectrum activity and low susceptibility to the development of resistance. To date, however, much remains unclear regarding their mechanism of action. Using functional assays (ATP leakage, cell viability, DNA binding) and super-high resolution structured illumination microscopy (OMX-SR) of fluorescently tagged polymers, we present evidence for a complex mechanism, involving membrane permeation as well as cellular uptake, interaction with intracellular targets and possible complexation with bacterial DNA. STATEMENT OF SIGNIFICANCE: This manuscript details the first study to systematically and directly investigate the mechanism of action of antimicrobial polymers, using super-resolution fluorescence imaging as well as functional assays. While many in the field cite membrane permeation as the sole mechanism underlying the activity of such polymers, we present evidence for multimodal actions including high cellular uptake and interaction with intracellular targets. It is also the first report to show competitive binding of antimicrobial polymers with bacterial DNA in a dose-dependent manner.

Interfacial Forces at Layered Surfaces: Substrate Electrical Double-Layer Forces Acting through Ultrathin Polymer Coatings.

Manipulating the surface properties of materials via the application of coatings is a widely used strategy to achieve desired interfacial interactions, implicitly assuming that the interfacial forces of coated samples are determined exclusively by the surface properties of the coatings. However, interfacial interactions between materials and their environments operate over finite length scales. Thus, the question addressed in this study is whether interactions associated with bulk substrate materials could act through thin coatings or, conversely, how thick a coating needs to be to completely screen subsurface forces contributed by underlying substrates. Plasma polymer layers were deposited on silicon wafer substrates from ethanol vapor, with identical chemical composition, ultrasmooth surfaces, and varying thicknesses. Using colloid-probe atomic force microscopy, electrical double-layer forces were determined in solutions of various ionic strengths and fitted using the Derjaguin-Landau-Verwey-Overbeek theory. For the thicker ethanol plasma polymers, the fitted surface potentials reflected the presence of surface carboxylate groups and were invariant with thickness. In contrast, for coatings <18 nm thick, the surface potentials increased steadily with decreasing film thickness; the measured electrical double-layer forces contained contributions from both the coating and the substrate. Theoretical calculations were in agreement with this model. Thus, our observations indicate that the higher surface potential of the underlying SiO2 surface can influence the interactions between a colloid particle and the multilayer structure if coatings are sufficiently thin. Such superposition needs to be factored into the design of coatings aimed at the control of material interactions via surface forces.

Engineering the Biointerface of Electrospun 3D Scaffolds with Functionalized Polymer Brushes for Enhanced Cell Binding.

Electrospun ultrafine fibers prepared using a blend of poly(lactide- co-glycolide) (PLGA) and bromine terminated poly(l-lactide) (PLA-Br), were surface modified using surface-initiated (SI) Cu(0) mediated polymerization. Copolymers based on N-acryloxysuccinimide (NAS) and a low fouling monomer (either N, N-dimethylacrylamide (DMA), N-(2-hydroxypropyl)acrylamide (HPA), or N-acryloylmorpholine (NAM)) were grafted from the fiber surface to impart surface functionality and to reduce nonspecific protein adsorption. Inclusion of the functional NAS monomer facilitated the conjugation of a nonbioactive cyclic RAD peptide and a bioactive cyclic RGD peptide, the latter expected to facilitate cell adhesion through its affinity for the αvß3 integrin receptor. A detailed analysis of the surface of the electrospun fiber scaffolds in nongrafted form compared to the surface functionalized state is presented. Characteristic amino acid peaks are observed for both conjugated RGD and RAD peptides. Cell culture experiments confirmed cell specific attachment mediated through the presence of the bioactive RGD peptide mainly at high surface density.

Long-Term Maintenance of Human Pluripotent Stem Cells on cRGDfK-Presenting Synthetic Surfaces.

Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.

Optimisation of grafting of low fouling polymers from three-dimensional scaffolds via surface-initiated Cu(0) mediated polymerisation.

Electrospun fibres represent a realistic implantable scaffold containing most of the structural three-dimensional (3D) characteristics of the extracellular matrix. However, as a result of their often synthetic nature, surface energy and chemistry, these scaffolds may adsorb a layer of non-specific proteins which can evoke a foreign body response. The precise surface modification of the scaffolds is challenging due to the complex geometrical and structural organization of the fibre meshes, that may limit the efficacy and completeness of approaches used. One flexible strategy that has gained attention is the use of reversible deactivation radical polymerisation (RDRP) techniques, which allow the creation of polymer brushes with controlled molecular weight, whilst retaining fibre morphology. In this study, protein adsorption was reduced with grafting of poly(N,N-dimethylacrylamide) (PDMA), poly(N-(2-hydroxypropyl)acrylamide) (PHPA) and poly(N-acryloylmorpholine) (PNAM) via surface-initiated (SI)-Cu(0) mediated radical polymerisation, from the surface of electrospun fibres prepared using a blend of bromine terminated poly(l-lactide) (PLA-Br) and poly(d,l-lactide-co-glycolide) (PLGA). Optimisation of the levels of Cu(i)Br, Me6TREN and the presence and concentration of a sacrificial initiator facilitated the grafting of well-controlled polymers brushes in less than one hour. Surface characterisation of the grafted scaffolds using X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS), and direct analysis of the molecular weight and polydispersity of polymer formed in solution during the reaction as well as the grafted polymer layer confirmed successful, controlled modification. Finally, protein adsorption experiments demonstrated the low adsorption properties of all polymer coatings with PDMA showing superior performance.

Surface modification of electrospun fibres for biomedical applications: A focus on radical polymerization methods.

The development of electrospun ultrafine fibres from biodegradable and biocompatible polymers has created exciting opportunities for biomedical applications. Fibre meshes with high surface area, suitable porosity and stiffness have been produced. Despite desirable structural and topographical properties, for most synthetic and some naturally occurring materials, the nature of the fibre surface chemistry has inhibited development. Hydrophobicity, undesirable non-specific protein adsorption and bacterial attachment and growth, coupled with a lack of surface functionality in many cases and an incomplete understanding of the myriad of interactions between cells and extracellular matrix (ECM) proteins have impeded the application of these systems. Chemical and physical treatments have been applied in order to modify or control the surface properties of electrospun fibres, with some success. Chemical modification using controlled radical polymerization, referred to here as reversible-deactivation radical polymerization (RDRP), has successfully introduced advanced surface functionality in some fibre systems. Atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT) are the most widely investigated techniques. This review analyses the practical applications of electrospinning for the fabrication of high quality ultrafine fibres and evaluates the techniques available for the surface modification of electrospun ultrafine fibres and includes a detailed focus on RDRP approaches.

One-Reactant Photografting of ATRP Initiators for Surface-Initiated Polymerization.

Self-initiated photografting polymerization is used to couple the polymerizable initiator monomer 2-(2-chloropropanoyloxy)ethyl acrylate to a range of polymeric substrates. The technique requires only UV light to couple the initiator to surfaces. The initiator surface density can be varied by inclusion of a diluent monomer or via selection of initiator and irradiation parameters. The functionality of the initiator surface is demonstrated by subsequent surface-initiated atom transfer radical polymerization. Surfaces are characterized by x-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM), and UV-induced changes to the initiator are assessed by (1) H NMR and gel permeation chromatography (GPC). This is the first time this one-reactant one-step technique has been demonstrated for creating an initiator surface of variable density.

Searching for new strategies against polymicrobial biofilm infections: guanylated polymethacrylates kill mixed fungal/bacterial biofilms.

OBJECTIVES: Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. METHODS: We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms. RESULTS: Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2ΔΔ mutant, we implicate matrix polysaccharide ß-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy. CONCLUSIONS: Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms.

Controlling integrin-based adhesion to a degradable electrospun fibre scaffold via SI-ATRP.

While polycaprolactone (PCL) and similar polyesters are commonly used as degradable scaffold materials in tissue engineering and related applications, non-specific adsorption of environmental proteins typically precludes any control over the signalling pathways that are activated during cell adhesion to these materials. Here we describe the preparation of PCL-based fibres that facilitate cell adhesion through well-defined pathways while preventing adhesion via adsorbed proteins. Surface-initiated atom transfer radical polymerisation (SI-ATRP) was used to graft a protein-resistant polymer brush coating from the surface of fibres, which had been electrospun from a brominated PCL macroinitiator. This coating also provided alkyne functional groups for the attachment of specific signalling molecules via the copper-mediated azide-alkyne click reaction; in this case, a cyclic RGD peptide with high affinity for αvß3 integrins. Mesenchymal stem cells were shown to attach to the fibres via the peptide, but did not attach in its absence, nor when blocked with soluble peptide, demonstrating the effective control of cell adhesion pathways.

Biomedical applications of polymers derived by reversible addition - fragmentation chain-transfer (RAFT).

RAFT- mediated polymerization, providing control over polymer length and architecture as well as facilitating post polymerization modification of end groups, has been applied to virtually every facet of biomedical materials research. RAFT polymers have seen particularly extensive use in drug delivery research. Facile generation of functional and telechelic polymers permits straightforward conjugation to many therapeutic compounds while synthesis of amphiphilic block copolymers via RAFT allows for the generation of self-assembled structures capable of carrying therapeutic payloads. With the large and growing body of literature employing RAFT polymers as drug delivery aids and vehicles, concern over the potential toxicity of RAFT derived polymers has been raised. While literature exploring this complication is relatively limited, the emerging consensus may be summed up in three parts: toxicity of polymers generated with dithiobenzoate RAFT agents is observed at high concentrations but not with polymers generated with trithiocarbonate RAFT agents; even for polymers generated with dithiobenzoate RAFT agents, most reported applications call for concentrations well below the toxicity threshold; and RAFT end-groups may be easily removed via any of a variety of techniques that leave the polymer with no intrinsic toxicity attributable to the mechanism of polymerization. The low toxicity of RAFT-derived polymers and the ability to remove end groups via straightforward and scalable processes make RAFT technology a valuable tool for practically any application in which a polymer of defined molecular weight and architecture is desired.