Hydrogel materialshave been employed as biological scaffolds fortissue regeneration across a wide range of applications. Their versatilityand biomimetic properties make them an optimal choice for treatingthe complex and delicate milieu of neural tissue damage. Aside fromfinely tailored hydrogel properties, which aim to mimic healthy physiologicaltissue, a minimally invasive delivery method is essential to preventoff-target and surgery-related complications. The specific class ofinjectable hydrogels termed self-assembling peptides (SAPs), providean ideal combination of in situ polymerization combined with versatilityfor biofunctionlization, tunable physicochemical properties, and highcytocompatibility. This review identifies design criteria for neuralscaffolds based upon key cellular interactions with the neural extracellularmatrix (ECM), with emphasis on aspects that are reproducible in abiomaterial environment. Examples of the most recent SAPs and modificationmethods are presented, with a focus on biological, mechanical, andtopographical cues. Furthermore, SAP electrical properties and methodsto provide appropriate electrical and electrochemical cues are widelydiscussed, in light of the endogenous electrical activity of neuraltissue as well as the clinical effectiveness of stimulation treatments.Recent applications of SAP materials in neural repair and electricalstimulation therapies are highlighted, identifying research gaps inthe field of hydrogels for neural regeneration. As an indicator of impact,the economic loss has been estimated at $800 billion in the UnitedStates alone.19 Biomimetic scaffolds, made frombiologically inspired materials, provide environmental cues that targetdesired biological mechanisms.254 Such biomimetic cues can be used to control cell and tissue behavior,promoting neural tissue regeneration and repair. These elements cantake the form of bioactive molecules and pharmaceuticals, as wellas mechanical and topographical cues for physical support.6 These tissue scaffold materials need to be carefullydesigned and tailored to elicit the desired cellular responses andthus provide a therapeutic effect.The overarching aim of tissue engineeringscaffolds is to use amaterial system to mimic the physicochemical properties of the naturaltissue milieu.26 A variety of hydrogel materialshave been investigated for neural applications, spanning from naturaltissue components to entirely synthetic materials.27 Biologically sourced materials including acellularized tissue andextracellular matrix-derived macromolecules such as collagen, chitosan,and hyaluronic acid have been used extensively. They are advantageousbecause they are nontoxic, cytocompatible, simple to obtain, and haveinherent bioactive cues, however biologically sourced materials carrya risk of immunogenicity and may be prone to batch-to-batch variability.28 Synthetic polymers present an alternative withsignificant benefits, including reproducibility and versatile tailoringthrough simple modifications of pendant groups. Common examples usedin tissue engineering constructs include poly(ethylene glycol) (PEG),poly (PVA) and poly(ethylene oxide) (PEO).31 However, these purely synthetic hydrogels lack critical biologicalcues, limiting their biomimetic properties.32 As such, tuning of the physicochemical properties and biofunctionalizationof these polymeric materials toward a more biomimetic material isoften necessary. Biologically inspired proteins or polymers are athird class of material that provides a higher degree of control incontrast to biological polymers, but being based on natural aminoacids (AAs) can be assembled to incorporate critical biological cues,such as adhesion sequences.34 These synthetic peptidescan be cross-linked into tunable, nontoxic, and biofunctionalizedhydrogels, making them a promising material choice for neuroregenerationapplications.37Hydrogel systems are themost commonly applied biomaterial forsoft tissue engineering. Hydrogels are ideal for these applicationsbecause of their structural and mechanical similarity to the extracellularmatrix components, their general cytocompatibility, and their capacityto provide biological cues.39 This was due to the need for material polymerization and implantdefinition prior to the surgery as a means of controlling the polymerstructure and structural features.41 The more recentdevelopment of minimally invasive and in situ surgical approacheshas fostered the development of injectable systems.42 These systems have found utility in neural repair, as theysupport localized treatment and minimize postsurgical complications,demonstrating versatility for translation to the clinic.20 Injectable materials permit the formation of a hydrogel in situ via the minimally invasive delivery of a hydrogelprecursor to the desired location. Once injected, the hydrogel canbe formed using a variety of physical or covalent cross-linking methods,including environmental stimuli such as temperature, pH and salt concentration.45 Both natural and synthetic polymers can be designed to be injectable,such as chitosan-based thermoresponsive hydrogels or injectable PEGpolymers.47 The combination of hydrogel precursor andmethod of polymerization will determine the final molecular arrangement,allowing for finely controlled macromolecular conformations.49Cellscaffolds are intended to physically support the surroundingtissue during regeneration. Historically, the scaffold shape and sizewere defined preimplantation, leading to surgical invasiveness andlong recovery periods.50 The design of self-assembling polymers requiresa precise understanding of chemical structures and molecular interactionsthat impact on the assembly mechanisms from monomer or macromonomerinto a hydrogel network.51 The addition of biofunctional groups must not chemically or structurallyinterfere with the self-assembling cross-linking mechanism of thepolymer, and simultaneously the mechanical and structural propertiesneed to be maintained within the physiological range.20 This complex design challenge requires versatile controlover the polymer chemical and structural composition. Among all materialtypes, peptide-based polymers offer the possibility to easily implementself-assembling mechanisms by mimicking natural aggregation processes,while maintaining the required physicochemical properties.54 The synthetic peptides that spontaneously assemble into orderednanostructures under physiological conditions are named self-assemblingpeptides (SAPs).57 One of the major advantages of SAPs among other materialtypes is their simple functionalization with adhesion molecules andtheir highly biocompatible components. SAP building blocks are effectivelysingle AAs, which are an important component of the physiologicalenvironment.55 Besides the simple synthesis,functionalization and property modification, these materials allowfor minimally invasive treatments, which are critical in neural injuryor disease.58The class of injectable materials termed self-assembling,offera thermodynamic advantage by exploiting spontaneous physical interactionsof the molecules in the environment, forming stable network microstructures.This review examines the recent developments in SAP systemsdesignedfor neural applications, including methods to tailor SAP propertiesto optimize their performance as neural scaffolds which can guideneural repair. Key design criteria are identified from an overviewof the physiological tissue properties, with the aim of replicatingthe main features of the neural environment within the biomaterial.Ways to control and tailor properties of SAP constructs, such as self-assemblingmechanisms, mechanical properties, topography, and bioactivity areconsidered as biomimetic cues through the lens of cell–materialinteractions. Furthermore, the incorporation of conductive scaffoldsand electrical stimulation within SAP constructs to promote neuralregeneration is assessed. Finally, the latest SAP-based applicationsfor neural regeneration are presented, to identify their advantagesand limitations, highlighting the latest technological advances andunmet clinical needs.259 Understanding the specific injury environment that a biomaterialis intended to address is critical to the successful development ofan injectable neural scaffold. The functionality and structure ofphysiological neural tissue relies on the synergy between a multitudeof specialized cell types and a complex microbiological milieu. Forinstance, the CNS and PNS have different responses to injury and varyin their potential for regeneration.63Cells need to sense specific biomimetic cues expectedfrom thenative ECM and healthy neural tissue to accomplish neural regenerationand repair. It is essential to consider these requirements for neuralrepair in the design of materials systems intended to address neuralinjury. Materials used for neural repair should therefore aim to mimicthe neural environment with finely tuned physicochemical propertiesengineered to interact with the target cell types and tissue features.64 Conversely, the injurysetting in the CNS triggers the reaction of microglia, astrocytesand oligodendrocytes, which inhibit regeneration and promote the creationof a glial scar.7 The two conditions presenta different biochemical environment, characterized by specific ECMcomposition, signaling cues and cell types. Design of a biomaterialimplant should consider all the relevant components and create a favorableenvironment for the proliferation, development and neurogenic behaviorof target cell types. Drugs and bioactive molecules can also be incorporatedwithin a material system for a multifunctional therapeutic approach.66 Biomaterials and in particularhydrogels may also be used as cell carriers in stem cell transplantsto control cell fate and promote neuroregenerative processes.67 The material cues for this applicationshould replicate the neural stem cell (NSC) niche, a biophysical microenvironmentthat regulates differentiation cues and cell fate.68 Cues toward neuronal lineage, as opposed to glial and epithelial,are preferred for an optimal integration with the endogenous nervoussystem.70After a peripheral nerveinjury, the distal segment of the axonundergoes an initial degeneration that inhibits growth in the initialstage, followed by the secretion of neurogenic signaling pathwaysby Schwann cells and the formation of growth cones for functionalnerve regeneration.72 Specific design criteria for material parameters and compositionshould be defined by considering the key components of the nativeneural milieu and their effect on cell behavior. Among all materialtypes, injectable materials require extremely precise tuning and characterizationof the biomimetic features postassembly, because the in vivo polymerizationdoes not allow for a preimplantation control of the material propertiesand self-assembling bioproducts. Fundamental material features suchas mechanical properties, degradation mechanisms, biochemical composition,structural features, and conductivity should be investigated in lightof both the physiological environment and the cell–materialinteractions to define effective injectable material properties andmodifications.The design of biomaterials targetingneuroregeneration should accountfor the complex host–material interactions for specific injuryenvironments, tailoring the cell interaction to the targeted tissuetype, diseases environment and cell type.2.127 Cell behaviorcan be directed toward neuroregeneration through the incorporationof bioactive cues within biomaterials.74 This material modification is exceptionally important in neuralapplications, given the low inherent low regenerative potential ofthis tissue type.75 The native ECM offersessential biochemical and structural cues to neural cells, which sensethe environment through adhesion molecules, termed integrins.80 Integrins are specialized adhesion receptors that interact withpeptide sequences present in the ECM and regulate cell–cellinteractions.81 They interact with thecell cytoskeleton and influence gene expression, proliferation, andsurvival through bidirectional signaling with the biochemical environment.84 It follows that the presence of integrin-binding factors is a paramountdesign requirement in biomaterials. Specifically, this includes ensuringcell adhesion through the presence of naturally derived materialsor the presence of biomimetic adhesion molecules.84The primary requirementfor neural repair is the presence of a biochemical environment thatsupports neural cell populations.87 Chondroitin sulfate influences neural plasticity and cell behaviorthrough sequences of sulfate groups on the GAG molecule backbone,conveying functional information through sulfation codes.89 In the case of the PNS, laminin,and collagen are fundamental ECM components for their role as Schwanncell regulators.90 It follows that ECMadhesion molecules are considered a powerful tool to direct cell behavior.84 In neural scaffolds laminin,collagen and hyaluronic acid are often selected as adhesion substratesin their natural or synthetic form.87 In particular,laminin-derived peptides in neural cultures are able to increase neuralcell migration, proliferation and differentiation toward neuronalfate.92 Short bioactivesequences of AAs involved in the adhesion signaling, termed bioactiveepitopes, are often exploited as adhesion cues in tissue engineering.The bioactive epitopes contained in laminin, collagen and fibronectinmolecules, including RGD, IKVAV, and YIGSR, are the most widely usedexamples93 , including chondroitin sulfate and hyaluronan.99 GFs are a widespread class of proteins that can stimulate cell growth,differentiation, and wound healing.101 Cell-binding of GFsactivate intracellular second messenger systems through cell surfacemembrane receptors that affect neural cell growth and differentiation.103 GFs are produced by healthy cell populations and can direct NSCdifferentiation toward specific cell types.74 Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),and tyrosine kinase (Trk) are important examples of a neurotrophicfactors involved in neural development which enhance neuronal differentiation.103 Other methods of biochemicalguidance include signaling molecules that drive gene cascades towardneural repair or differentiation.105 For instance,the delivery of a molecule dubbed TTK21 was recently proven to promotespinal cord regeneration and sprouting of sensory and motor axonsthrough epigenetic reprogramming.105 In addition,the neural chemical signaling molecules neurotransmitters are knownto influence neural plasticity and are involved in strengthening neuralconnections and glial cell stimulation.107 GFs and bioactive molecules can be incorporated inthe material system to enhance neural regeneration or direct cellfate, and their effect can be tailored for regenerative or drug deliveryapplications.Aside from adhesion peptides,other bioactive molecules such asgrowth factors (GFs), cytokines, and signaling molecules are consideredeffective cues acting through regeneration-related molecular pathwaysin the CNS and PNS.2.2110 More comprehensive properties of the brain, spinal cord, and PNSare presented in 111 Transmembrane proteins, primarily integrins, are intrinsically mechanosensitiveand affect cell behavior and growth depending on substrate stiffness83 , NSCdifferentiation can be directed toward a substantial neuronal subpopulationas opposed to astrocyte and oligodendrocyte populations.116 Koser et al.114 showed that axon lengthand degree of spreading varies with substrate stiffness. Softer substrateswere shown to encourage more exploratory growth, better suited forsynaptic formation, whereas stiffer substrates promoted faster, straighter,and more parallel growth of axons.117 Astiffness range above 200 kPa can lead to apoptotic activity and reducedviability of in vitro neural cultures.93 This phenomenon has also been observed in the clinical setting,where damage to the CNS causes the formation of scar tissue, or glialscar, which dramatically increases the stiffness of the tissue, leadingto neural loss and cell death.8 Zhong et al.118 have performed a comprehensive review of mechano-sensingunder 2D and 3D environments.The mechanicalproperties of neural tissue vary depending on tissue type and location.In general, the brain has a low stiffness and presents viscoelasticproperties, whereas nerves and the spinal cord show higher tensilestrengths due to the alignment of the nerve fibers.ffness83 1. Bindin120 However, an engineering challengecan be identified in the design of injectable materials. Self-assemblymechanisms can be affected by variable physiological conditions anddelivery methods, such as temperature, chemical composition of thetarget site, or injection speed.43 Thesefeatures can cause difficulties in achieving precise mechanical propertiesto ensure physical support to the cells.47When designing a biomaterial scaffold for neural repair, the mechanicalproperties should be based on physiological ranges, and design criteriashould specifically target substrate stiffness to support neuron survivaland direct cell behavior toward regenerative processes. Modificationsof the elastic or compressive modulus can be implemented in materialsystems to match the target tissue features with relatively simpleapproaches that have been detailed in the literature.124 Neural cells interactwith their environment by degrading as well as producing ECM.30 Neural tissue physiological remodelling is a fundamental processin healthy tissue environments, involved in tissue turnover, synapticplasticity and neural repair. Enzymes known as matrix metalloproteinases(MMPs) are responsible for ECM degradation and remodelling and promotetissue growth and differentiation.125 Neuronsand glia secrete degradation MMPs and contribute to ECM remodellingin physiological conditions, brain injury, and other brain disorderssuch as cancer.129 Abnormal ECM dynamics, commonly presentin injured or pathological tissue, may also cause imbalances in cellbehaviors leading to immune and inflammatory response activation,which encompass the initial stage of spontaneous neural repair. Forexample, after spinal cord injury (SCI), the molecules released fromdamaged ECM can trigger and amplify the inflammatory response. Thesubsequent alterations of the ECM structural and chemical compositionaffect cell migration, communication, and survival toward a spontaneousregenerative response.85 These mechanismsaffecting tissue remodelling can be replicated to provide both endogenousand exogenous cells with a substrate to degrade while proliferatingand secreting new ECM.130 A balance between providingmechanical support and allowing space for tissue growth is a centralrequirement to achieve a physiological cell response to the biomaterialand avoiding adverse responses.131Importantly, mechanical support provided to encapsulated cellschanges dynamically with material degradation, which can be tunedto match natural tissue growth.21 A trade-off between controlled degradation and biocompatibilityshould be considered.36 A high degradation rate can leadto the accumulation of chemical degradation products, which in turncan encourage glial scarring and immune/foreign body response.133 Thus, the material composition and degradable chemical bonds shouldbe engineered to match the natural tissue degradation rate of 2–6weeks.134 Degradation is typically dueto hydrolytic or enzymatic degradation.136 Functional groups such as MMP cleavable peptide linkages can beinserted into a biomaterial to match the degradation with local cellproliferation and metabolic activity.137 It is important to note that the degradation rate in vitro and in vivo can vary considerably because of thechanges in environmental conditions.138Theideal scaffold provides initial mechanical and biochemicalsupport to cells, and its degradation rate should match the ECM formationsuch that it allows for the regeneration and growth of the new tissue.2.3139 In the PNS, aligned nervefibers are organized in fascicles depending on function, displayinga hierarchical architecture,140.141 The nerve sheath, composed of myelin and connectivetissue, surrounds and insulates nerve fibers.141 The spinal cord has a similar aligned architecture, showingascending and descending neurons organized in bundles, around 8–60μm in size123.142 The brain structure is more homogeneous, with the whitematter composed of aligned myelinated nerve fibers and the gray matterconsisting of cell bodies and unmyelinated axons, with highly anisotropicstructures.139 The brain ECM includes perineuronalnets (PNNs), which show lattice-like chondroitin sulfate structuresaround subpopulations of neurons. They act as growth and migrationinhibitors to maintain the tissue structure.75 Replicating these physiological structures can be advantageous fora scaffold’s efficacy, given that the tissue architecture candirectly affect cell behavior and function.139 Indeed, aside from sensing the substrate’s stiffness, surfaceand adhesion receptors can also respond to the architecture and topographyof the environment.63The micro-and macroscale structures of neural tissue are linked to their physiologicalfunction.148 It is important to note that historically the majority of in vitro cell studies have been performed in 2D cultures.150 However, the native neural milieu and its physicochemical featuresare 3D. This implies a significant difference in the way cells areaffected by environmental cues. The spatial distribution of the cuesis more homogeneous and this affects cell attachment and shape towarda more biomimetic model.153 As a result, 3D spatial featuresof a construct can influence the neural cell response, and in vitro3D cultures created by encapsulating cells within a biomaterial area preferable method for replicating the neural environment.149 3D architectural cues can be introduced intothe material system as topographical cues to neural cells. Topographicalcues include every spatial feature and physical modification of themicroenvironment, spanning from fibrous structures to roughness ofthe surface.155 Curtis et al.156 have reviewed how cellssense physical features of the environment at the nano- and microscalesuch as physical patterning, roughness, pits, grooves, and fiber alignment.Surface patterning and roughness affect cell attachment and migration,158 while chemical patterning modifies cell morphology.159 Aligned topography is found to be among themost effective in neural tissue regeneration, due to their polarizedmorphology, which mimics physiological patterns in neural tissue.163 Human NSCs are shown to differentiate toward the neuronal lineagewhen exposed to aligned microscale patterns, and neurite outgrowthcan be enhanced by contact guidance.166 For example, dorsal root gangliacells increase the maximum length of their neurites by 82% when exposedto core–sheath nanofibers.167 Baraneset al.168 showed that nanotopographiesaltered gene expression profiles of primary neurons isolated frommedicinal leaches, upregulating axon-guidance signaling pathways,synaptogenesis and synaptic regulation, resembling the behavior ofinterconnected neurons. Human embryonic stem cells (hESCs) can bedifferentiated into a neuronal lineage by exposing them to an alignedridge pattern, without the need for other differentiation-inducingagents.143 Similarly, human induced pluripotentstem cells (hiPSCs) can be differentiated into neuronal lineages whenexposed to aligned microgrooves.169 Thisproperty can be exploited as a powerful method to control and tunethe development of a neural progenitor cell population, and guideits growth at the same time.171 This cellular responseis highly desirable for neural regeneration, and methods to createa material that elicits this cellular response in clinical applicationsare of utmost interest.172The spatialarrangement of micro- and nanoscale material features can influencecell adhesion, spreading, alignment, and morphology which in turncan alter cell behavior and gene expression.173 Specifically,the porosity and pore size of the material should be tuned to allowfor molecular diffusion while providing a stable structure for cellgrowth and proliferation.174 In neural applications,the pore interconnectivity is essential for neurite growth, with adesirable porosity of 90% and a suitable pore size pore size rangingfrom 10 to 100 μm.177Micro- and nanoscalestructures can also influence local homeostasisby affecting the accessibility of soluble nutrients, ions and molecules,as well as tissue vascularization.2.4178 Endogenous electricfields are known to be present in neural development and would healing.180 Spontaneous activity in the CNS plays a role in the assembly ofdeveloping neural circuits, and axon regrowth is promoted by the electricalpotential physiologically generated in the wound environment.179 Endogenous electrical signals consist of polarizedion transport within the biological tissue, which influences cellmembrane potential and electrophysiological state.181 The conductive properties of different types of neural tissue arepresented in Neural cellbehavior and growth can be substantially impacted by electrical cues,which are a widespread strategy for neuroregeneration treatments suchas nerve repair.188 In the context of neuroregeneration, neuronal guidancethrough biomimetic electrical signals is a powerful tool to repairnerve and spinal cord injuries.192 The electrophysiological stateof the stem cell niche is known to promote differentiation towardneural lineage and increased neural proliferation.190 The use of electrical cues in tissue engineering is extensive andspontaneous electrical potentials are a central element for neuraldevelopment, thus the conductivity and electrochemical propertiesof scaffold materials used for neural repair are worthy of consideration.195 An ideal material will support the endogenous or exogenous electricfield propagation to favor neural regeneration.194 In the context of SAPs, it is essential to ensure the compatibilityof the self-assembling physiochemical mechanism with the propagationof electrical signals.196 Alternatively, electroactivescaffolds can be developed to actively promote electrical stimulationor exposure of cells to electric fields.189Signaling pathways influencing the cell cycle, ion channel expression,and other gene cascades leading to proliferation, migration, and differentiationare activated by electrical activity.The reviewed design criteria cover an extensive range of materialproperties and relative cell–material interactions involvedin neuroregeneration mechanisms 2. Bioact360 Facilitating topographicalcues via injectable materials is challenging as it typically requires in situ formation of structural elements. Self-assemblingmaterials enable the formation of various topographies upon injectionin vivo due to their responsiveness to local environments. Therefore,careful design of the material can lead to control over physiochemicalproperties in order to achieve a scaffold that meets the criteriafor neural regeneration.197 Self-assemblyis governed by supramolecular chemistry as it relies on non-covalentforces between molecules. It is therefore important to understandthe forces that govern the self-assembly process in order to tunethe assembled structures and their properties for a specific application.Methods have been developed to tune the topographical, mechanical,bioactive and conductive properties of self-assembling materials.The application of these methods to SAPs can be tailored to createa biomimetic and effective material support.Scaffolds for tissue engineering neural repair should minimizeinvasiveness and provide topographical, structural, biomechanicaland biochemical support for neural regeneration. Various attemptshave been made using synthetic or biological materials; however, allthese material modifications need to be considered with regards totheir potential impact on the physicochemical properties of the biomaterialconstruct.198 These intermolecular forcesare dominated by hydrogen bonds, electrostatic interactions, hydrophobicinteraction, and π–π interactions. Therefore, externalstimulations to trigger self-assembly include the effect of pH, temperature,ionic charge and concentration as well as various other triggers suchas enzymes and phototriggers. Different intramolecular driving forcesand external stimulations can guide the self-assembly of polymer systems.These interactions have been extensively reviewed and are summarizedin 202Noncovalent interactionsbetween molecules are the driving forcefor the spontaneous formation of organized structures, a process calledself-assembly that occurs readily in nature at various length scales.A variety of molecular driving forces can be used to create self-assemblysystems.204It is important to consider these known driving forceswhen tailoringthe topographical, mechanical and electrical properties of SAPs forneural regeneration. Strong interactions such as ionic forces andcoordination bonds require consideration in the design of a systemthat will self-assemble in the conditions found within the nervoussystem. Weaker interactions such as van der Waals electrostatic andhydrophobic interactions, H-bonding, and π–π stackinghave strong influences on the self-assembled morphology, mechanicalproperties and bioactivity of SAPs. A balance between these forcescan create molecules that will self-assemble into fibers in aqueousconditions but form a hydrogel when strong ions are introduced, thusenabling control over their gelation and subsequent material properties.To date, various types of self-assembling molecules in physiologicalenvironments have been explored ranging from synthetic small molecules,proteins, peptides, nucleic acids and hybrids as detailed in 209 Most of these self-assembling molecules are in part driven by theinterplay of hydrophobic and hydrophilic forces. For example, syntheticblock copolymers comprised from alternating hydrophobic poly and hydrophilic poly(ethylene glycol) segments form a self-assemblinggel in aqueous conditions and this has been shown to support neuronaldifferentiation when loaded with growth factor releasing microspheres.210 Similarly, the RADA16-I is a peptide consistingof 16 AAs with alternating hydrophobic and hydrophilic residues. Thisdrives its self-assembly in aqueous environments into a stable β-sheetstructure.19 Alternately, Watson–Crickbase pairing in DNA can be utilized to form self-assembling nanotubesof DNA segments, which can be functionalized with peptide sequencesthat promote neural differentiation.211 A common example of hybrid biomolecules are peptide amphiphiles(PAs), which consist of a hydrophilic peptide head, often followedby a β-sheet forming sequence, which is then capped with a hydrophobicsegment. This leads to hydrophobic collapse in aqueous conditions.200 The hydrophobic tail can consist of alkyl chains,aromatic molecules such as Fmoc or other functional molecules.213 Nucleic acids and peptide or peptide amphiphiles are an ideal materialbecause of their inherent low immunogenicity and versatile biofunctionality.57 PAs can easily be synthesized on both small scales for experimentalstudy and large scale for application in the clinic.217 Peptides can also be functionalized with synthetic moleculesin order to create amphiphilic molecules that self-assemble into avariety of different morphologies including fibers which promote thedifferentiation and elongation of neural stem cells, serving as atopographical guide for their growth.219 These self-assemblingsystems can be utilized to make materials across multiple length andspatial scales.207 Some of the most commonmorphologies are linear, trigonal, and cyclical structures, whichthen self-assemble to form various secondary and tertiary structuresas illustrated by The chemical structureof these molecules allows control over size,shape, charge, and surface properties while maintaining low cytotoxicity.3.1232233 developed a PA that wouldtransition from molecules dispersed in solution to micelles or nanofibersbased on pH. A reduction in pH of 0.8 transformed micelles into nanofibers.233 This pH and concentration responsiveness isillustrated in The nanotopographyof self-assembled structures can be modulatedby varying the molecular structure or the environment in which theself-assembly occurs. More specifically, techniques such as changingthe molecular design, electrostatic capping, pH, self-assembly moleculeconcentration or solvents have all been used to control the formationof micelles, β-sheets, α-helix, nanobelts, and membranes.234 By varyingthe pH it was shown that a flat amphiphilic peptide nanobelt couldbe transformed into a grooved nanobelt with parallel nanochannels.228 Interestingly, a concentration-dependent modulationof morphologies was also demonstrated.228 Different structures including a split nanobelt with bristle morphologyand twisted nanoribbons were achieved by reducing the concentrationof PA molecules in the aqueous solution.235The morphology of a self-assembly structure canalso be fine-tunedby pH as shown by Cui et al.236 ultrasonication,electric fields, or external force fields.238 For example, Zhang et al.239 used shear force from the injection of an aqueousPA into an ionic solution to form a noodle-like hydrogel of alignedpeptide nanofibers. This aligned PA was later functionalized withIKVAV and RGDS bioactive epitopes and shown to promote aligned neuriteoutgrowth in P19 mouse neurons.218 It alsoresulted in the formation of synapses and spontaneous electrical networkformation after 2 weeks in culture with hippocampal neurons.218Self-assembling fibers can be hierarchically organized insupramolecularcrystals which can be aligned using various methods such as acousticfields, pressure, magnetic fields,241 Co-assembly and AA modification can also changedimensions and sizes of fibrous aggregates, fostering the formationof 1D or 3D networks.243 These techniques can be harnessedto create nanotopographies that can promote neural regeneration. Co-assemblycan also be used to incorporate bioactive epitopes into the fibersin order to control cell fate.244Co-assembly is the incorporation of twoor more distinct buildingblocks that self-assemble to form a structure, similar to the coassemblyof proteins in nature. The combination of distinct components allowsfor the development of novel functional properties, and the tuningof supramolecular morphology and bioactivity as well as the physicochemicalproperties of the hydrogel. Various methods exist to obtain coassemblyharnessing aromatic interactions, enzymatic action, electrostaticinteraction, chemical stimuli, or electromechanical stimuli.3.2245 concentration,247 coassembly, cross-linking,248 and solvent/ionsinteractions the mechanical properties can be tailored.252 The mechanical properties of SAPs that have beenachieved using these methods can be found in 249 showed that by modifying peptide concentrationand sequence charge of an oligopeptide the elastic modulus of theresulting hydrogel can be varied across 2 orders of magnitude from2–200 kPa. Shear thinning and self-healing properties werealso demonstrated through reassembly, which are of interest for insitu placement.249 Shantanu et al.111 explored the effect of varying gel stiffnesson hippocampal cells. By varying the strength of the β-sheetinteractions PAs with stiffness of 22.9 and 7.3 kPa were designed.111 Hippocampal neurons were subsequently culturedon peptide coated surfaces and it was found that the stiffness ofthe substrate greatly affected astrocyte density and neuronal maturation.111 Stiffer substrates led to an astrocyte density10 times higher than softer substrates, while neuronal density was30% lower on stiffer substrates compared to soft self-assembled fibers.111 This demonstrated that varying stiffness allowsfor control over the differentiation of neural cells.111 Furthermore, the effect of stiffness on neuronmaturation, classified by morphological criteria, was apparent afteronly 20 h in culture.111 Interestingly,softer peptide amphiphile scaffolds showed faster maturation of neurons,which was not dependent on the presence of KDI or RGDS epitopes.111Self-assembling structureshave tunable mechanical properties. Byvarying sequence charge, branching,118 Degradationof self-assembling materials can be tuned by varying the molecularstructure. For example, the incorporation of sequences that can becleaved by MMPs has led to the degradation of β-sheet fibrillarmaterials.258 However, the expression of MMPs is hard to control in vivo. An alternativethat has been investigated is the incorporation of ester bonds intoself-assembled gels, rendering the degradation dependent on pH andwater accessibility, a more predictable in vivo process. Collier etal.259 showed that by incorporating glycolicacid (Glc) within the peptide segment of an Fmoc-F-RGD SAP resultedin a linear degradation profile over 60 days. Placement of the Glcsegment was critical, as substituting the glycine in the RGD sequenceresulted in greatly reduced bioactivity of the adhesion epitope.259 Placement of the Glc segment next to intactRGD sequences permitted hydrolytic degradation without compromisingthe bioactivity of the RGD sequence.259 The stiffness of the degradable gel was around 1.5 kPa.259 When coassembled with and Fmoc-diphenylanilinepeptide which has a stiffness of 30 kPa, a range of stiffnesses wasobtained depending on the ratio up to a stiffness of 13 kPa for a20:1 ratio of Fmoc-FF to Fmoc-F-Glc-RGD.259 Rho et al.260 showed that secondary hydrophobicinteraction near the core of cyclical peptides can stabilize the peptidebonds without compromising on solubility in aqueous conditions.Scaffold degradationallows cells to remodel the ECM, thus improvingmigration and viability.3.3153 An overview of recentlyexplored bioactive cues incorporated in SAP materials is presentedin 138 These molecules are necessary for cell survival,migration, and differentiation and cell behavior can be influencedby modifying the scaffold’s adhesion cues.261 Decellularized ECM materials or purified single ECM componentscan be engineered as injectable natural scaffolds to preserve thephysiological chemical environment.262 Hyaluronan, methylcellulose,chitosan, and fibrin among other materials can be used to design insitu forming biomaterials for neural repair and drug delivery.264 However, such materials can present batch-to-batch variability,and tuning their composition or material properties can be challenging.42 Synthetic SAPs offer the possibility of multiple functionalizationswith targeted molecules and epitopes in predefined concentrations.130 Thus, bioactive ECM components can be includedin self-assembling material design maintaining constant biochemicaland physical conditions.265Awide range of bioactive cues have been incorporated within biomaterialsintended for neural repair. SAPs offer the possibility of multifunctionalizingthe material system, by simultaneously incorporating bioactive moleculesin the peptide sequence and within the scaffold structure. The versatilityof their biofunctionalization is a major advantage in the field ofneural scaffold materials.58 Neural bioactive peptide motifs tested for usein biomaterials are usually derived from the amino-acidic sequenceof the neural cell adhesion molecule (NCAM), fibrin, laminin, andfibronectin.58 Aside from the universal adhesionmolecule RGD laminin-derived epitope, IKVAV can be considered themost popular example in neural engineering for its role in neuralstem cell differentiation and glial scar reduction, especially whencombined with SAPs such as RADA-IKVAV and PA sequences.268 Epitope peptides can be synthesized directlyat any site of the SAP backbone sequence or chemically ligated asa postsynthesis modification, in a linear or branched fashion.272 Solid phase peptide synthesis is one of the most common techniques,chosen for the relatively simple method and versatility.275 Investigation on the effect of differentbioactive epitopes, epitope density and exposure are possible becauseof the highly controllable chemical structure of SAPs and precisematerial purification methods.275 Silva et al.275 synthesized a peptide-amphiphile material thatassembles into nanofibers at physiological pH and functionalized itby chemically binding the IKVAV epitope at one extremity of the sequence motifs, achieving an increase in primaryNSC proliferation and neural differentiation, whereas maintaininga stable and precise GF delivery and concentration. GFs can also beencapsulated into the polymer network through physical bonds whichbreak upon hydrogel degradation.283 Finally, Gelain et al.283 achieved clinically viable GF release profiles incorporating negativelycharged AA sequences to the SAP RADA16-I. The positively charged basic-fibroblastcytokine (βFGF) electrostatically interacted with the SAP terminus,allowing for a gradual release, which increased NSC proliferation.283 GFs can also be combined with adhesion epitopesusing different incorporation methods, to enable effective deliveryto the tissues. Rodriguez et al.282 synthesizedthe SAP Fmoc-DIKVAV as a single peptide chain and subsequently incorporatedglial cell line derived neurotrophic factor (GDNF) by physically entrappingthe molecule within the hydrogel upon gelation. This allowed for adual effect on NSC differentiation and proliferation by the IKVAVepitope and NGF, which improved the regeneration effect of a NSC transplantin Parkinson’s disease mice models  forbrain diseases and functional electrical stimulation (FES) for spinalcord injuries.301 However, although these devicesare designed to replace lost function, without scaffold support, thereis minimal capacity for neural tissue regeneration. In fact, the implantationand presence of a rigid device can result in further neural cell loss.The employment of scaffold materials within bioelectronics applicationshas been gaining attention over the past decade, including the useof soft polymeric electronics for implants, neural interface coatingmaterials, and drug delivery systems.303 These technologieshave revealed both the potential for organic conductors applied inelectrical stimulation and the need for scaffold materials that arecompatible with electrical stimulation.305 Coupling bionic devices with tissue engineered scaffoldsis an emergent area where conductive SAPs may find application. However,it is essential to ensure the compatibility of the self-assemblingmechanism, which is often driven by electrostatic interactions withthe propagation of electrical signals.196Electrical stimulation is a powerfultool for neural repair. Thetherapeutic effect of stimulation is supported by a range of treatmentstargeting diverse injury settings and applications.310 and ionically porous materials have been used to ensure that cellsare effectively exposed to electric fields.189 Incorporation of conductive materials into self-assembling scaffoldshas been investigated as a method of providing cell scaffolds withconductive elements. Carbon based nanomaterials such as nanotubes(CNT) and graphene have been explored to confer electroactivity toscaffolds. Although they demonstrate good conductivity and polymercomposites have been designed with appropriate mechanical properties,the regulatory pathway for new materials and in particular carbonnanomaterials has hindered their clinical translation.312 Conductive polymers (CPs)313 have alsoemerged as a potential solution due to their high charge injectioncapacity and ionic conductance.316 CPs are characterized by alternating single and double bonds alongthe backbone, termed π-conjugation, which cause a delocalizationof electrons. Some of the most commonly used CPs for in vivo studiesare poly (PEDOT), polypyrrole (PPy), andpolyaniline (PANI).60 The primary disadvantageof CPs are their poor mechanical stability and limited conformationalcontrol.318 The addition of bioactive cues to CPs to improve cell attachmentand proliferation can also have a significant impact on the polymerproperties, preventing the possibility of a multifunctional biomimeticscaffold from a bulk CP.319Electroactivescaffolds have been developed to actively promoteelectrical stimulation320 Conductive hydrogels (CHs)have been developed pursuing this concept and applied to flexiblebioelectronic applications.322 The coupling of these conductive materials to self-assembling hydrogelshas also been investigated.325 Relevant examples of natural and syntheticself-assembling materials compatible with electrical stimulation orthat possess intrinsic conductive properties can be found in 326 However,this conductivity has been shown to be highly dependent on the secondaryand tertiary structures,327 making it difficultto tailor these systems for neural repair applications.To improvethe scaffold electrical properties while maintainingcytocompatibility and mechanical tuneability, researchers can introduceconducting elements such as CPs or CNTs to softer and more tunablematerials.4.1328 For example,Fmoc functionalization of SAPs have been shown to aid self-assemblyby enhancing π–π stacking.329 In peptide-based hydrogels, aromatic compounds are usedas gelators, helping form hydrogels that are mechanically stable andbiocompatible for various applications such as drug carriers or antifouling/antibacterialgels.330 Aromatic compounds are used tocap the N-terminus in solid phase peptide synthesis, and can thereforeeasily be integrated into the material synthesis.328 The incorporation of aromatic compounds and oligomers in self-assemblingmolecules has been explored to make conductive materials for a widevariety of applications such as electronics, optics, optoelectronics,photovoltaics, magnetic and piezoelectric devices, sensors, drug releasinghydrogels, and catalysts.339Self-assembly ofsmall molecules enables a bottom-up control of material properties.Aromatic compounds are mostly incorporated into hydrogels for biomedicalapplications because they enhance the formation and stability of hydrogelsin self-assembling systems.340 Supramolecular electronics have studied theassembly of π-conjugates into electronic nanowires.341 Many of these self-assemblies occur in organicsolvents, which inherently limits their application to neural regenerationwhere a key design criterion is in situ formationvia injectable preproducts. Furthermore, the stability and degradationof electroactive scaffolds in physiological environments is not wellunderstood. Despite these current limitations, the approaches takenin these associated fields that employ self-assembly techniques demonstratethe tuneability and feasibility of developing 3D conductive self-assemblingnetworks for neural repair.343 For example, highlyconductive BTBT amphiphiles are commonly formed in organic solvents,but recently, it has been demonstrated that self-assembly of thesemolecules can be achieved in aqueous conditions, in a first step towarda tissue engineering application.344 Interestin these π-conjugated peptides for biomaterial applicationswill continue to grow because of the tuneability and nontoxic natureof self-assembling electroactive molecules.346 Understanding the various methods that have been used to tailorthe electronic properties of π-conjugated oligomer self-assemblingsystems is crucial to determining their compatibility for neural tissueengineering.The field of nanoarchitectonics has studied the hierarchicalorganization of self-assembling molecules into functional layers,sensors, bioactive components, and artificial living systems. Conductivelayers have therefore been developed by incorporating aromatic moleculesand small linker molecules to various self-assembling molecules.349 Peptide π-conjugates areoften composed of an AA region, a CP region, and sometimes a hydrophobicalkyl tail region. Modifying the various components of this molecularstructure can affect several material properties. Lehrman et al.350 showed that by varying the AA side chain ofpeptide thiophene structures allows for control over the resultingnanostructure. By substituting AA of varying size and hydrophobicity,it was demonstrated that π–π stacking and hydrogenbonding both contribute to self-assembly but are also competitiveforces.350 It was suggested that the controlof nanostructures arises from the optimization of the balance betweenπ–π stacking, intermolecular hydrogen bonding,and attractive van de Waals forces.350 Changingthis balance results in different morphologies including flat structures,spiral sheets, or nanotubes.350 Panda etal.351 also showed that by varying thealkyl spacer length between the peptide and the aromatic componentof the self-assembling structure the chirality of a triblock β-sheetfiber could be tuned. All-atom molecular simulations were subsequentlyused to design peptide sequences to control peptide chirality andelectron delocalization properties.352 Peptidechirality affects the conformation and morphology of the resultingstructure and is therefore of great interest for bioactivity.353 The core oligomer length was also shown toinfluence the phase behavior and morphology of self-assembled structures.Different oligomer lengths can lead to the formation of high-aspect-ratiofiber networks or disordered aggregates.354 Alternatively, varying the solvent has been shown to change theself-assembled morphology.356 Doping of the conjugatedstructures has also been shown to alter the morphology of self-assembledmolecules. Mushtaq et al.335 showed thatPEGylated tetra (aniline) self-assembled into spherical nanostructures.It was shown that these nanostructures can be doped using HCl, whichincreases their size.335 These structureswere found to be electroactive through cyclic voltammetry and UV–visspectroscopy investigations.335 These polymerswere shown to have excellent cytocompatibility when injected intothe liver of rats.336To develop a biomimetic scaffold understandinghow the incorporationof π-conjugated systems affects topographical, mechanical, andconductive properties is key. Varying the molecular structure hasbeen shown to tune the secondary structure and morphology of aromaticself-assembled molecules.357 incorporated tetra(aniline) into an SAP nanofiber and demonstratedthat neurite outgrowth and differentiation of PC12 cells was enhanced6 days after induction with NGF. It was demonstrated that this conductiveSAP upregulated the phosphorylation level of the ERK1/2 pathway relativeto the nonfunctionalized peptide in an investigation of the upstreampathways of NGF induced neural-like differentiation.357 Although these studies show the significant promise ofSAPs, many do not characterize the SAP electrical properties in physiologicalconditions.Finally, ithas been shown that biological benefits can be achievedfrom these electrically modified SAPs. Guler et al.368 demonstrated the hierarchical organization of self-assembling π-conjugatedfibers into aligned macroscopic domains. Aligned macroscopic domainsof optoelectronic structures were fabricated using a shear-flow assemblyas shown in 368 As discussed above, topographicalcues from self-assembled aligned fibrous networks have been shownto guide neurite outgrowth, which is particularly applicable to mimickingspinal cord architecture for regeneration after SCI. Wu et al.369 studied the effect of combining topographicalcues with the photoconductive polymer poly(3-hexylthiophene) (P3HT)on neurite outgrowth. The growth and differentiation of PC12 cellswas assessed on a homogeneous P3HT film, self-assembled P3HT nanofibers,and electrospun P3HT/poly(e-caprolactone) (PCL) microfibers with andwithout light irradiation.369 This enabledan understanding of the effect of electrical stimulation due to thephotoconductive polymer in conjunction with different topographies.369 The electrical stimulation provided by theLED irradiation was shown to consistently promote increased neuriteoutgrowth on all topographies and the self-assembled nanofibers promotedthe longest neurite outgrowth.369The hierarchical organization of self-assemblingπ-conjugatedfibers is of interest for neural tissue engineering since combiningtheir conductive properties with topographical cues could lead tosynergistic effects on neural cells. Aligned fibers are of interestfor both spinal cord and peripheral nerve regeneration as they mimicthe topography found in vivo. Wall et al.370 studied the influenceof varying peptide sequence on mechanical and electrical propertiesand found that varying the length of AA side chains varied the topography,mechanical, and electrical properties of the formed gels. It was demonstratedthat the storage modulus could be increased from 3 to 20 kPa, withsheet resistance increasing from 5 to 17 kΩ sq–1 and secondary structures varying from α- to β-helix.Varying AA sequences changed the local conformation and stacking ofπ-conjugates.370 Interestingly, thesteric effects of the larger aliphatic tails or aromatic groups werefound to vary intermolecular electronic coupling within the nanostructuresas well as stacking, which was correlated to a reduced sheet resistance.370 This demonstrates the tuneability of self-assembledπ-conjugated peptides for neural tissue engineering. Thurstonet al.371 investigated the effect on theelectronic properties further through molecular modeling and densityfunctional theory calculations to show that smaller AAs favor linearstacking within the peptide dimer, and this improves the delocalizationof electrons. This confirmed that varying the AA sequence changesintermolecular forces and charge transport properties and is thereforean important tool to consider when designing a tissue engineeringscaffold for neural regeneration.It is important to understandhow these modifications to molecularstructure will affect the conductivity of the self-assembled π-conjugates.Ardoña et al.362 The conductive polymer segments can also becovalently cross-linked along the fiber axis to increase conductivityby adding free conductive polymer to the solution. Blatz et al.372 explored this idea by functionalizing a peptidewith EDOT–OH. The EDOT-modified peptide was polymerized witha 1:3 molar ratio of additional EDOT–OH in organic solventas shown in 372 Conductivity in the order of 10× 10–4 S cm–1 were reported,but it was hypothesized that doping the structure could greatly increaseits conductivity.372 Murphy et al.364 synthesized a library of 12 tetrapeptides andfunctionalized them with EDOT–OH, subsequently polymerizingthem with a 1:1 molar ratio and EDOT–OH in aqueous solutionsand doped the gel with pTSA.364 This yieldedgels with conductivities in the order of 1 × 10–2 S cm–1.364 Conductivityof the conjugated systems can also be improved by incorporating dopantswithin the molecular structure of self-assembling systems.373 Yang et al.374 createda 3D nanostructured CH for application as a pseudocapacitor. Ionicand electronic conductive properties of PPy and PANI functionalizedwith different molecules that act as both gelators and dopants wereinvestigated.374 These molecules self-assembleinto conductive fibers as shown in 374 Conductivities as high as 3.3 S/cm were obtained for thePPy-TB molecule shown in 374 This hydrogel wasdesigned for energy storage applications, and it is biocompatibilityhas not been explored but trypan blue is known to be toxic.375 However, this is a good demonstration of theuse of dopants to increase conductivity in self-assembled π-conjugatedsystems as other dopants are readily used in the synthesis of CPsfor neural tissue engineering.316 Anotherstrategy to increase the conductivity is the coassembly of differentself-assembling molecules.241 The coassembly ofelectron donors and acceptors has been shown to increase conductivity.For example, to create a 1D nanowire that self-assembles in aqueousmedia, Khalily et al.377 coassembled n-typeand p-type short peptide-chromophore conjugates. The coassembly showedincreased conductivity relative to either the n-type or p-type fibersalone. The n/p-co-assembled nanowires are approximately2400 times more conductive that the n-type wires, and are 10 timesmore conductive than the p-type alone.377Several methods have beenexplored to enhance the electron transportof self-assembled fibers. Nanofibers have been used as a templateto guide the self-assembly of conductive polymers, with additionalCP added to the self-assembly dispersion before gelation and oxidation.378 showed that variations of the AA side chainresidues at various locations along the AA side chains led to changesin π-stacking. π-stacking has been shown to have profoundeffects on the conductivity of self-assembled structure metal–organicframeworks,380 we can therefore infer thatconductive peptide packing will be affected by the AA sequence leadingto changes in conductivity. This is further reinforced by Lee et al.’s381 recent report of very high conductivity ina self-assembly system composed of a π-chromophore core flankedby an alkyl spacer and pentapeptide on both sides. Unexpected mineralizationof KCl along the glutamic AA in HCl vapor deposition was obtainedfollowing KOH treatment, which is thought to have led to proton dopingalong with very strong packing and stability.381 This method yielded conductivities as high as 1800 S cm–1 when incorporating alkyl spacers between the peptidesand π-conjugation.381 Obtaining suchconductivity values demonstrates the potential for the applicationof molecular self-assembly to form conductive networks.Understanding the relationshipbetween molecular structure andπ-stacking is important in the development of conductive self-assembledsystems containing π-conjugates. It is therefore interestingto note changes in π-stacking of chromophore self-assemblies.Varying the AA sequence of a PA has also been shown to tune chromophorepacking and their resulting photophysics. Tovar et al.4.2200 This option confers precise temporal controlover material properties and polymerization, fostering the developmentof cutting-edge applications such as stimuli-responsive drug delivery.206 This technology provides clear advantages in the field of neuralrepair, where the sensitive and complex environment requires temporallyand spatially precise interventions. Material features such as bioactivecues, wettability, and protein absorption, as well as assembly anddisassembly could be controlled by electrical stimulation,204.205 For instance, bioactive biotin moleculescan be reversibly exposed depending on the surface potential,200 and the presence of an electric voltage canmaintain drug-loaded nanoparticles in an assembled configuration tocontrol drug delivery.206 It is also knownthat the natural hydrogel chitosan reversibly self-assembles via electrodepositionof films which are physically cross-linked.208 Similarly, the redox reaction triggered by electrical stimulationof polydopamine (PDA) coatings have been shown to promote cell spreading,proliferation, and differentiation on a titanium electrode.209 This class of materials termed “switchable”can be used to implement highly controllable electrically mediatedtherapies, allowing for temporal and spatial control of bioactive,topographical, electrochemical, and structural cues to neural cells.Although the properties mentioned are representative of the wide rangeof possibilities offered by switchable materials, today they onlyserve as a proof of concept toward an innovative vision for SAP materials.The uses of electricalstimulation can be targeted not only to neural tissue but also towardspecific SAP material features. The possibility of finely tuning thestimulation parameters has promoted the concept of electrically responsivematerials.5216 In the PNS, SAPs have been investigated for treatment of crush andresections of sciatic nerves,11 cavernousnerves,382383 and facial nerves.219 Recently, Richteret al.219 compared SAP performance to anautograft (using the resected nerve segment) for the regenerationof the facial nerve after a 7.5 mm resection. A hollow collagen tubewas filled with an aligned PA and neural regeneration was assessedby electrophysiological stimulation and recording across the nerveresection site.219 It was found that thePA showed similar performance to the autograft which is an impressiveoutcome because autografts are considered the gold standard in treatment.219 This PA did not present any bioactive epitopes,but presents evidence that the regenerative potential of SAPs couldmatch autografts and lead to improvements in functional recovery.This is further supported by a 10 mm sciatic nerve resection studyled by Yang et al.384 Functional recoverysimilar to that of an autograft was demonstrated by using a SAP functionalizedwith both IKVAV and RGI, which mimics both laminin and BDNF.384In vivo applications of SAPhydrogels have shown great promisefor neural regeneration. Multiple studies have shown that SAPs elicitminimal inflammation and scar formation while promoting vasculatureformation, axonal regrowth, and synaptic formation.388 In one of the most recent applications to spinal cord regenerationthe properties of self-assembling systems were leveraged in orderto create a synergistic scaffold. Xiao et al.389 have combined topography with bioactivity and drug release.RADA16 SAP containing FGL  was tailoredto release Taxol for spinal cord injury in a rat model.389 Taxol has been shown useful in vitro but has difficulty crossing the BBB and therefore requires localizeddrug delivery. Following a T9 contusion, the SAPs were injected intothe lesion site and rats were evaluated up to 8 weeks after injury.The rats injected with a Taxol-loaded scaffold showed the most functionalrecovery with a ranking of 15 on the BBB scale, cell infiltration,and neurite extension across the lesion, as well as reduced glialactivation and inflammation, and reduced cavity formation.389 This is an example of the advantage of tailoringSAPs to control multiple properties critical to regeneration of thenervous system.In the CNS treatment of spinal cord injurywith SAPs has shownreduced inflammation, cavitation, and scar formation. The promotionof axonal growth and guidance, vascularization, and functional recoveryhas been observed in animal models.390 Cell-loaded, drug-loaded, or bioactive injectable SAPs are a strategyfor restoring brain tissue and function.58 For example, a SAP functionalized with the laminin epitope IKVAVwas found to promote the proliferation and differentiation of endogenousNSCs and to improve the learning and memory impairment in an Alzheimer’sdisease mice model.267SAPs are also an emerging option to accomplishbrain regenerationin the context of traumatic brain injury and neurodegenerative diseases.14 andbridging the gap between regeneration therapies and electrical stimulationdevices is becoming a necessity.391 Smartconductive materials are emerging for bioelectronics applicationswhere the versatility of SAPs could be harnessed for functional electro-neuralinterface therapies promoting neural regeneration. Chromophore andelectroactive peptides have been studied for in vivo drug deliveryand tracing and imaging of tumors and have demonstrated good biocompatibilityand stability.393 However, more work needs tobe done on the application of electroactive self-assembling scaffoldsfor neural regeneration. Material development of biocompatible andelectrically tunable SAPs needs to be undertaken with specific attentionto biocompatibility of the components and self-assembly mechanismfor injection in vivo. Additionally, the stabilityand degradation of electroactive scaffolds in physiological environmentsneed to be understood to characterize its effect on stiffness andelectroactivity of the scaffold. The properties of these novel materialscan then be tailored to suit the material properties of neural tissueenabling the understanding of the degree of synergistic effects possiblethrough utilizing multiple biomolecules, topographies, and coassemblies.Ultimately the translation of materials developed in the lab not onlyto in vivo models that are known to have regenerativecapacity but to the clinic where the systemic patient disease or injurystate can impact cell–material interactions needs to be investigated.Although thereis a plethora of studies on self-assembly systemsfor neural regeneration both in vivo and in vitro, no complete functionalrecovery has been observed to date in long peripheral nerve gap injuriesor severe central nervous system injuries. Complementary electricalstimulation approaches such as DBS are among the leading treatmentsfor late stage neurodegeneration6Self-assembling materials provide a significantopportunity fordeveloping next-generation neural regeneration scaffolds. There arechallenges in designing injectable biomimetic SAPs, but because oftheir bottom-up design, these self-assembled structures are highlytailorable. Varying molecular structure and intramolecular interactions,as well as ionic concentrations, can ensure that bioactivity, mechanical,and topographical properties can meet neural tissue engineering regenerationcriteria. SAPs offer significant improvement by filling the heterogeneousinjury cavities, promoting cell survival, migration, and differentiationas well as axonal growth into through the lesion site. However, inCNS lesions and extensive PNS damage, SAPs do not completely restorefunction to the injured tissue.Future developments of SAPsmust consider the design of materialsthat provide a combination of biomimetic cues to promote the regenerationof neural tissue through provision of multiple physical and biochemicalcues. Historically, SAPs and indeed other hydrogel materials for neuralregeneration have not been tailored through combining all of thesebiomimetic cues but rather focused on a single or two desired properties.More complex combinatorial systems that better replicate the naturalneural milieu may address the current limitations faced by tissueengineering approaches to neural regeneration. Specifically, it isthought that electrical properties will be critical to supportingthese electroactive tissues. Various methods exist to incorporateelectrical activity into self-assembling systems for the developmentof nanoelectronics; however, few studies have assessed their applicationto neural regeneration. Some studies have investigated the applicationof electroactive or chromophore functionalized self-assembling systemsfor responsive in vivo drug delivery which suggests the potentialbiocompatibility of electrically functionalized SAPs. However, keylimitations in the current research include minimal characterizationof electroactive properties and a lack of understanding of how thesecomponents impact degradation and subsequent long-term electroactivityof the scaffold.


The COVID-19 pandemic has a substantial impact on essential immunization activities.Disruption to mass vaccination campaigns increase risk of VPD resurgence.Catch-up campaigns are necessary to limit existing shortfalls in vaccine uptake.Guidance to mitigate these effects continues to evolve.The current COVID-19 global pandemic continues to impact healthcare services beyond those directly related to the management of SARS-CoV-2 transmission and disease. We reviewed the published literature to assess the pandemic impact on existing global immunization activities and how the impact may be addressed. Widespread global disruption in routine childhood immunization has impacted a majority of regions and countries, especially in the initial pandemic phases. While data indicate subsequent recovery in immunization rates, a substantial number of vulnerable people remain unvaccinated. The downstream impact may be even greater in resource-limited settings and economically poorer populations, and consequently there are growing concerns around the resurgence of vaccine-preventable diseases, particularly measles. Guidance on how to address immunization deficits are available and continue to evolve, emphasizing the importance of maintaining and restoring routine immunization and necessary mass vaccination campaigns during and after pandemics. In this, collaboration between a broad range of stakeholders  and clear communication and engagement with the public can help achieve these goals. Widescale infectious disease outbreaks exert considerable direct and indirect effects on a societal, economic and public health level. At present, we remain in the midst of the coronavirus disease 2019 (COVID-19) global pandemic due to severe acute respiratory syndrome-coronavirus-2 [SARS-CoV-2] since its emergence in late December 2019  implemented as local or more widespread national mass vaccination campaigns), providing direct protection and a broader indirect protection through herd immunity. Immunization service disruption threatens to reverse hard-won progress on VPDs ; the higher the R0, the higher the vaccine coverage necessary for infection control at the population level) [R0 of 12–18 and 12–17 respectively) and where necessary vaccine coverage for herd immunity is higher (e.g. 92–94% for measles) than infections with lower transmission rates such as diphtheria (R0 of 6–7) [R0 is generally considered lower, with correspondingly lower herd immunity thresholds required to stop community transmission [These general observations raise concerns that much of the public health gains realized by immunization will be lost. Estimates indicate that each year immunization programs prevent 2.7 million cases of measles, 2 million cases of neonatal tetanus, 1 million pertussis cases, 600,000 cases of poliomyelitis, and 300,000 diphtheria cases . Much ofn level) . This is of 6–7) ,84. In csmission .While addressing the COVID-19 pandemic is an immediate concern, we must remain vigilant of other pathogens. An early modelling study (from a pan-Africa perspective) estimated that continuing routine immunization would avert a far greater number of lives lost due to VPDs compared to every single potential death due to SARS-CoV-2 averted with suspension of routine immunization services . ConcernGuidance on immunization activities have evolved throughout the COVID-19 pandemic, and continues to do so. While initial guidance was to halt planned mass vaccination campaigns (which had some impact) , currentSimilarly, routine immunization should continue ,47, inteSince the COVID-19 pandemic was declared in March 2020, we have learned many important lessons that should inform future pandemic preparedness plans to help mitigate the many risks and challenges we have faced, including minimizing the impact on routine immunization programs. Vaccine hesitancy should continue to be addressed, as its impact is not limited to pandemic vaccines but continues to extend to routinely recommended vaccines ,103. We Finally, we do not need to wait for the start of a future pandemic to continue our efforts and exploring new approaches on raising awareness of the public health benefits of vaccination, and while most of the disruptions we describe relate to childhood immunization, a broader perspective also applies. As the world population gets older, the overall positive impact of adult vaccination on health and wellbeing raises important questions and opportunities to make sure this twenty first century “older” adult population will also translate into an “older and healthy” population . An onliSupplementary Figure 1.The current COVID-19 pandemic has exerted a substantial toll beyond the direct effects of SARS-CoV-2 morbidity and mortality; chiefly social and economic disruption and in its impact on routine healthcare services including routine immunization activities. Immunisation rates have fallen substantially during the pandemic, placing individuals and communities at greater risk of VPD. Addressing existing shortfalls and strengthening routine services is essential to avoid routine immunisation becoming another casualty of the pandemic. Collaboration between a broad range of stakeholders  allied with clear public communication and engagement to restore confidence towards increasing routine vaccine uptake and necessary catch-up activities can help achieve these goals. A plain language summary of our observations is presented in Click here for additional data file.


Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal. Renal transplantation is the established treatment of choice for kidney failure, as it confers both the highest survival and the best quality of life compared to other renal replacement therapies . DespiteThe induction of tolerance, i.e. the lack of destructive immune responses to a transplanted organ in the absence of immuno-suppression, could potentially overcome both of these limitations. Tolerance in kidney transplantation can be functionally defined by stable renal function and absence of histologic, immune and molecular signs of rejection on a kidney biopsy obtained after complete withdrawal of immunosuppression for at least one year. Spontaneous tolerance is unfortunately a rare and unpredictable event that has been described in a small minority among the patients who choose to discontinue their immunosuppression, who retained graft function despite complete withdrawal of immunosuppression .Among the different methods used to induce tolerance in animal models of kidney transplantation, few have been successfully translated to clinical application. Those protocols that have succeeded in patients entail combined kidney and bone marrow transplantation (CKBMT) as a strategy to induce chimerism, a state wherein donor hematopoietic cells engraft into the recipient bone marrow at a level sufficient to be detected by conventional (as opposed to sensitive PCR-based) methods.Three centers have developed clinical CKBMT protocols, one of which has so far succeeded in achieving tolerance only in the HLA-identical transplant setting . InvestiOnly two strategies have succeeded in effectively inducing operational tolerance across HLA barriers so far. As HLA mismatches are commonly present in solid organ transplantation, in this review we will discuss the features of these regimens and the novel mechanistic insights offered by recent studies in the field.Animal Studies. More than 60 years ago, Main and Prehn used bone marrow infusion following administration of high-dose, lethal total body irradiation (TBI) to achieve skin allograft tolerance in recipient mice. In this experimental setting, semiallogeneic but not isogenic bone marrow infusion consistently permitted donor-specific skin graft acceptance  and expand IL-10-producing Tr1 cells  in mice treated with high TBI doses. Murine FC include a population of CD8αr1 cells –17. Thesr1 cells  and haveClinical Protocols. Investigators from Northwestern University utilized a non-myeloablative conditioning regimen that achieved durable full donor chimerism in humans, attempting to exploit the engraftment-promoting and immunosuppressive effect of FC  and were successfully weaned off immunosuppression after one year from transplant. These subjects showed significantly better kidney function compared to matched controls receiving conventional immunosuppression. Two graft losses due to opportunistic infections were recorded in the first year after transplantation, and one tolerant recipient died due to sepsis. Studies in mice have highlighted that full donor chimeras are somewhat immunoincompetent , 23 due Animal Studies. Mixed chimerism defines a state wherein, unlike full donor chimerism, the host hematopoietic system is not completely destroyed and replaced by the donor’s, and hematopoietic cells of both the recipient and the donor coexist in the bone marrow.via intra-thymic negative selection of alloreactive T cell clones, mediated by both donor- and recipient-derived antigen-presenting cells  . This meng cells , 29 and ng cells . Durableng cells –33. Pering cells –38 and ang cells , 39. Theng cells , 41. Notng cells , 43  in patients with and without hematologic malignancies. Differences between these regimens have been reviewed in detail elsewhere  experienced chronic rejection or glomerulonephritis recurrence, which led to reintroduction of immunosuppressive drugs and ultimately resulted in graft loss more than 10 years after transplantation. Of note, these patients were successfully retransplanted with conventional immunosuppression, and there were no significant opportunistic infections in any of them.Initial studies used a non-myeloablative conditioning regimen that included cyclophosphamide, the anti-CD2 T cell-depleting monoclonal antibody siplizumab and thymic irradiation (TI) Table 1 In parallel with early host T cell recovery, 9 patients unexpectedly developed severe acute kidney injury. Renal histology was consistent with engraftment syndrome, entailing capillary endothelial injury with vascular leak and lympho-monocytic infiltrating cells in peritubular and glomerular capillaries. Renal function normalized in all but 2 recipients, one of whom experienced graft loss due to acute humoral rejection as a consequence of preformed DSA that were undetectable on a pre-transplant ELISA, but were subsequently confirmed by Luminex. In the other patient, acute kidney injury was initially misdiagnosed as rejection and was treated with higher doses of tacrolimus, which triggered thrombotic microangiopathy. Finally, one patient developed severe cellular rejection after a pyelonephritis episode following immunosuppression withdrawal. Protocol biopsies (at 2-8 years) in tolerant subjects showed either completely normal histology or minimal alterations, including focal glomerular basement membrane duplication and mild podocyte foot process effacement .An additional protocol was tested at MGH based on further observations from studies conducted in non-human primates , 51. ComInvestigators at the Samsung Medical Center initially used a nearly identical protocol to those outlined above, but the anti-CD2 monoclonal antibody siplizumab was substituted with ATG due to local unavailability , 53. To The mechanism that underlies tolerance to kidney allografts associated with full-donor chimerism hypothetically involves central tolerance of donor T cells to donor antigens, with donor progenitor cells migrating to the recipient thymus, differentiating into antigen-presenting cells and finally mediating negative selection of “self”-reactive donor T cell clones. Bulk functional assays, including mixed-lymphocyte reactions (MLR) and cell-mediated lympholysis (CML), demonstrated donor-specific hyporesponsiveness in tolerant patients. However, the same effect was observed in recipients who exhibited only transient chimerism and developed rejection after immunosuppression withdrawal , suggestde novo GVH tolerance might be characterized by a combination of clonal deletion, anergy and regulatory T cell-mediated mechanisms. However, GVH tolerance has not been demonstrated in these patients and the inclusion in the infused product of mature donor T cells that eliminate host hematopoiesis suggests that an ongoing GVH reaction may occur, which has culminated in GVHD in several patients. Whether or not GVH reactions in patients without overt GVHD results in thymic injury and failure to negatively select host-reactive T cells, as reported in murine models  and on a minimal fold-expansion , while computational methods were used to account for sorting errors  with evidence of peripheral proliferation, possibly recent thymic emigration and, in one patient, conversion from conventional T cells , Immune Tolerance Network contract (N01 AI15416) and Immune Tolerance Network Award (UM1 AI109565).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The survival rate for metastatic osteosarcoma has not improved for several decades, since the introduction and refinement of chemotherapy as a treatment in addition to surgery. Over two thirds of metastatic osteosarcoma patients, many of whom are children or adolescents, fail to exhibit durable responses and succumb to their disease. Concerted efforts have been made to increase survival rates through identification of candidate therapies via animal studies and early phase trials of novel treatments, but unfortunately, this work has produced negligible improvements to the survival rate for metastatic osteosarcoma patients. This review summarizes data from clinical trials of metastatic osteosarcoma therapies as well as pre-clinical studies that report efficacy of novel drugs against metastatic osteosarcoma in vivo. Considerations regarding the design of animal studies and clinical trials to improve survival outcomes for metastatic osteosarcoma patients are also discussed. Osteosarcoma is the most common primary cancer of the bone. Its emergence is typically driven by mutations in the TP53 and RB1 genes of osteoblastic cells or their precursors in the extremities, such as the tibia or femur ,3. OsteoThe 5-year survival rate of osteosarcoma was as low as 20% when it was originally defined in the 1950s . The autMost osteosarcoma patients currently receive doxorubicin, cisplatin and high-dose methotrexate (referred to as “MAP”) as first-line therapy, alongside limb-sparing surgery to remove the primary tumor. This regimen is associated with a 5-year survival rate of around 70% for localized disease. MAP is also administered as a first-line regimen to patients with metastases at diagnosis, although it is less effective for these patients: between 10% to 40% of them survive for 5 years or more after diagnosis.Retrospective studies have underscored the utility of metastasectomies for patients with pulmonary metastases, including repeated operations to remove any subsequently detected lung lesions ,10,11. EA wide variety of systemic therapies have been explored for patients with relapsed or refractory osteosarcoma. Regorafenib (discussed below) is the only drug whose use as a second-line therapy was uniformly recommended by the National Comprehensive Cancer Network on the basis of high level (category 1) evidence, with high-dose ifosfamide plus etoposide and sorafenib receiving recommendations as second-line regimens based on less conclusive evidence . Factors that portend particularly poor prognoses for patients with osteosarcoma metastases include: distant bone lesions (compared to the more common lung metastases) ,16,17,18At diagnosis, around 20% of osteosarcoma patients have detectable metastases. The lungs make up 90% of metastatic sites, with other bones accounting for a further 8% to 10% ,23. ManyThis review specifically summarizes pre-clinical and clinical trial data relating to novel therapies for metastatic osteosarcoma, to document progress towards improving outcomes for these patients. The standard neoadjuvant and adjuvant MAP chemotherapy regimen for patients with localized disease is also administered to patients with metastatic disease, before and after attempting to remove lesions by metastasectomy . Various substitutions and additions have been explored, but unfortunately, none have yielded increased survival rates for patients with metastatic osteosarcoma.Newer generations of anthracycline, such as pirarubicin, that reportedly have better tumor uptake and reduced toxicities have been examined as a substitute for doxorubicin . A trialPemetrexed is an inhibitor of thymidylate synthase and has a broader range of action than methotrexate. A phase II trial was conducted to evaluate the efficacy of pemetrexed as a single agent in 32 metastatic osteosarcoma patients . One patCisplatin’s efficacy is hindered by dose-limiting nephrotoxicity. Trials have evaluated the efficacy of carboplatin, a less toxic platinating agent , as a reOne phase II trial evaluated the addition of ifosfamide to doxorubicin and cisplatin for metastatic disease but found no clinical benefit over standard treatment . AlthougThe use of the topoisomerase II inhibitor etoposide as an additional agent, coupled with MAP plus ifosfamide, has been evaluated in two trials for metastatic osteosarcoma. A phase II/III trial evaluated the efficacy of this combination in 43 patients newly diagnosed with metastatic osteosarcoma . The twoA phase II trial examined the efficacy of the water soluble camptothecin analogue topotecan, after promising data from in vivo testing on osteosarcoma xenografts ,39. TwenA phase II trial examined the efficacy of high-dose thiotpeta  with autologous transplantation as an adjuvant to standard chemotherapy in 22 patients with relapsed metastatic osteosarcoma, but reported no significant clinical benefit compared to the 22 control patients who received chemotherapy alone . GemcitaL-alanosine is an inhibitor of de novo adenine synthesis. It was evaluated in 65 carcinoma and sarcoma patients, including seven osteosarcoma patients, three with metastases. Participants’ tumors featured methylthioadenosine phosphorylase deficiency , a deterThe bisphosphonate zoledronic acid inhibits bone resorption by suppressing osteoclast differentiation, and also exerts anti-cancer activities through incompletely defined pathways . ZoledroMany osteosarcoma patients initially respond to chemotherapy, but a challenge in maintaining remission is balancing efficacy with the myelosuppressive activity of these treatments . AutologThe relatively high level of infiltrating lymphocytes in osteosarcomas compared to other sarcomas ,54 have After encouraging results from treating non-metastatic osteosarcoma patients with the immune modulator liposomal muramyl tripeptide (mifamurtide) , additioThe combination of recombinant interleukin 1α and etoposide, which was documented to provoke anti-tumor activity by lymphoid cells , was triA high proportion of osteosarcomas, particularly pulmonary metastases, express programmed cell death protein-1 ligand (PD-L1). This suggests that metastases may be especially sensitive to PD-1 inhibitors such as pembrolizumab ,65. SeveMany osteosarcoma patients have human epidermal growth factor receptor 2 (HER2) positive tumors, which formed the basis of a trial evaluating HER2-specific chimeric antigen receptor modified T-cells (CAR T-cells) against HER2-positive sarcomas . UnfortuAlthough the previously described immunotherapies failed to improve patient outcomes, a prospective study of metastatic osteosarcoma patients who received MAP in addition to IL-2 and lymphokine activated killer (LAK) cell reinfusion yielded more promising results . Of 27 pReceptor tyrosine kinases represent a broad set of targets, some of which are often overexpressed in osteosarcomas, giving rise to a novel approach of treating metastatic osteosarcoma with tyrosine kinase inhibitors (TKI) . Poor osOncoLar is a somatostatin analogue that inhibits IGF-1 production, which can suppress the growth of osteosarcoma in vitro  and metaRobatumumab is an IGF-1 receptor inhibitor that showed promising pre-clinical activity in subcutaneous patient-derived xenografts  and was Many of the TKIs currently being evaluated have multiple targets, including receptor and intracellular tyrosine kinases that drive pro-tumorigenic pathways, such as angiogenesis and proliferation . Data frRegorafenib is a multitarget TKI that has been tested for treating metastatic osteosarcoma across two randomized phase II clinical trials. The REGOBONE trial compared regorafenib versus placebo in patients with metastatic osteosarcoma who had failed to respond to chemotherapy. Regorafenib significantly delayed disease progression. Of 26 eligible treated patients, two had partial responses and 15 stable disease with the remainder showing disease progression . SARC024Sorafenib is another multitarget TKI that has been well studied in osteosarcoma patients. In a phase II trial of 35 patients with metastatic or advanced osteosarcoma, sorafenib treatment led to a reduction in tumor burden in 14% of patients, but only 29% had not progressed within 6 months of commencing treatment . A subseCabozantinib is an inhibitor of VEGFR2 and MET and was studied in 43 osteosarcoma patients, of which 39 had pulmonary metastases, in a phase II clinical trial . SimilarOther drug classes have been trialed for metastatic osteosarcoma based on promising pre-clinical data, but have yielded generally disappointing outcomes. Glembatumumab vedotin is an antibody-drug conjugate that targets an anti-mitotic agent to cells expressing glycoprotein non-metastatic B protein, which is overexpressed in most osteosarcomas . When teDespite the numerous trials conducted to evaluate novel therapies for metastatic osteosarcoma, the long-term survival of patients remains unchanged . Many studies have been published evaluating the efficacy of candidate treatments for metastatic osteosarcoma using mouse models. These models are often generated by orthotopic or subcutaneous injection of human or murine osteosarcoma cells into mice, which can metastasize to the lungs over the course of an experiment . The preIt is sobering to reflect that the previously described clinical trials failed to recapitulate the exciting efficacy published using pre-clinical animal models. This mismatch may reflect inadequacies of these animal models, in addition to a publication bias favoring reporting of positive outcomes, which presumably would be more prominent in pre-clinical than clinical studies.Directly seeding osteosarcoma cells to the lungs by intravenous injection can be used to establish a metastatic model without a primary tumor  ,149,150.Although these models are a powerful tool, it can be difficult to determine if a drug is active against established metastases unless researchers commence treatment after metastases have formed. Authors evaluating therapies such as 3-hydroxyflavone, euxanthone, CXCR4, Timosaponin, TRAIL, Panobinostat, mycophenolic acid, zoledronic acid, rapamycin, parthenolide or quercetin began treatment prior to the injection of cells or shortly afterwards ,157,158.Studies in which authors waited at least one week post-cell injection prior to commencing treatment make a more compelling case for a drug’s efficacy against metastases, as the cells would have presumably arrived at the lungs and begun forming pulmonary tumors. The mTOR inhibitor rapamycin improved the survival rate of xenograft and isograft mice bearing pulmonary metastases compared to those treated with vehicle . The oncIdeally, researchers would confirm the presence of metastases in mice prior to starting treatment. Very few studies have been published in which the authors detected established metastases in the lungs of mice by microscopy (in pilot experiments) and/or in vivo imaging before beginning treatment. Nasarre et al. observed additive cooperation between PD-1 inhibitor and an antibody targeting Secreted Frizzled-Related Protein 2 (SFRP2) to reduce the number of metastases on the surface of mouse lungs by commencing treatment mice with the antibody in conjunction with a PD-1 inhibitor eight to twelve days after cells were intravenously injected . The proAs an alternative to mouse xenografts and isografts, dogs present a useful model for evaluating new therapies for spontaneous metastatic osteosarcoma. Canine osteosarcoma shares many characteristics with human osteosarcoma, including immune infiltration, microenvironment, genetics and presentation of metastatic disease ,169. AltOf the clinical trials currently being conducted for metastatic osteosarcoma, almost half are evaluating immunotherapies such as mifamurtide, IL-2 or PD-1 inhibitors . TyrosinDespite intensive efforts to improve the outcome of patients diagnosed with osteosarcoma, including those with overt metastases, the survival rate for patients reported in clinical trials evaluating new therapies has only modestly increased at best. It seems the long-sought substantial improvements in patient survival will require additional basic biology research and development of drugs engaging relevant molecular targets, followed by well-designed animal experiments and phase II clinical trials, hopefully leading to the identification of promising candidate therapies for evaluation in phase III trials. Optimal clinical trial design will hasten progress towards this goal.Mineralization associated with osteosarcomas limits the utility of radiographic imaging for monitoring tumor responses to neo-adjuvant treatment ,174. HisThe most meaningful endpoint for clinical trials of osteosarcoma treatments is overall survival (OS). In practice, however, progression-free survival (PFS)  is more commonly used as a primary endpoint in phase II trials. Concerningly, while PFS and OS data correlated tightly in trials involving Ewing sarcoma patients , PFS wasWhether phase II trials should randomize patients to experimental therapies versus placebo is controversial. Some experts contend that single-arm trials with early endpoints can be informative and efficient, as historical benchmarks obviate the need to split relatively scarce patients between two treatment groups. The “New Agents for Osteosarcoma Task Force” devised a structured approach  to selecIn addition to providing insight into the potential utility of new therapies, clinical trials that incorporate predictive biomarker assays may reveal avenues for further research that may ultimately improve outcomes for patients. Understanding the physiological, cellular and/or biochemical factors that allow even a small proportion of patients to respond to a particular drug may facilitate personalized treatment approaches based on the features of patients’ primary tumors or metastases. Biomarkers were explored to predict patient responses to ridaforolimus, but none of the eight markers assayed varied between patients who did or did not respond to treatment . This maA study comparing whole genome and exosome sequences between osteosarcoma patients’ primary tumors and their pulmonary metastases revealed that metastases had a much higher tumor mutational burden and genomic instability . MetastaSeveral clinical trials of PD-1 inhibitors for carcinomas suggest that tumor mutational burden (TMB) may be useful for predicting patient response to immunotherapy ,192,193.The paucity of better-than-expected survival outcomes in osteosarcoma clinical trials summarized above suggests that a dramatic improvement in outcomes for the majority of metastatic osteosarcoma patients will probably require targeting of a novel process or molecule, distinct from those engaged by agents used in clinical trials to date. Hopefully, ongoing pre-clinical research will uncover such game-changing novel targets. Until/unless those approaches bear fruit, extending lifespans for some osteosarcoma patients may hopefully be realized by assembling a panel of therapies that exhibit efficacy in subsets of patients, coupled with development of biomarker assays to enable tailoring of treatments to individual patients.