Some classes of behaviors, including gambling, Internet gaming, and sexual behaviors, may lead to compulsive engagement for a minority of individuals. In extreme cases where individuals may feel unable to control these behaviors without external influence, these behaviors may be considered non-substance or behavioral addictions. Many such behaviors may occur predominantly online, such as gaming, social media, shopping, and pornography, and may be driven by constant accessibility via smartphone and other mobile device technologies. The diagnostic criteria for gambling disorder and Internet gaming disorder (IGD) in the DSM-5 are similar to substance use disorder, referring to symptoms of withdrawal and tolerance, continued use despite negative consequences, and loss of control over the activity. However, some behaviors such as compulsive buying and compulsive sexual behaviors do not have specific diagnostic categories in DSM-5. Many of these behaviors, including emerging online behaviors, will continue to be the subject of discussion among international authorities, such as the World Health Organization (WHO), including calls for more research evidence on behavioral addictions. This Research Topic presents diverse papers on neurobiological evidences of behavioral addictions, encompassing gambling disorder, Internet-based disorders, including Internet gaming disorder and smartphone addiction, and compulsive sexual behaviors.Hou et al. reported that changes in synaptic ultrastructure may be associated with withdrawal anxiety in alcohol dependence.The field of behavioral addictions is continually seeking to identify and understand the important neurobiological mechanisms that drive repetitive, maladaptive behaviors. Animal studies of substance addiction, for example, may help to guide research on the neurobiological mechanisms underlying behavioral addictions. Alcohol withdrawal-anxiety syndrome refers to symptoms that have been shown to depend on molecular and cellular adaptations that lead to persistent, long-term plastic changes in transcription, translation, and synaptic morphology. However, the molecular mechanism underlying the anxiogenic effects of ethanol withdrawal require further study. In the first paper in this Topic, Cho et al. used a rodent version of the gambling task (rGT) to examine how impulsive action and impulsive choice are differentially influenced by difference in age at exposure  to rGT in rats. The results indicated that impulsive action and choice are distinct aspects of impulsivity, which are differentially influenced in rats by the age at the first exposed to gambling task.Impulsivity is considered an important feature associated with the development of addictions. Wang et al. reported that increased insular cortical thickness correlated with symptom severity in individuals with IGD. In another study, Lee et al. reported that subregions of the anterior cingulate cortex, another key node of the salience network, formed different functional connectivity patterns in subject with IGD with comorbid depression.One of the major neural networks that play a crucial role in behavioral addiction is the salience network, which mediates the “switching” between neural networks to guide appropriate responses. Alterations in the salience network have been implicated in directing aberrant salience to stimuli associated with addiction, resulting in craving and impaired control over addictive behaviors. Hong et al. reported that the SS allele of 5HTTLPR genotype group showed greater FC within the DMN and salience network, and between these networks, compared to the SL + LL allele group. The results suggest that the short allele of 5HTTLPR may increase FC within the DMN and salience network, which may subsequently aggravate impulsive Internet game play in patients with MDD.Problematic Internet game play is often accompanied by major depressive disorder (MDD). Depression seems to be closely related to altered functional connectivity (FC) within (and between) the default mode network (DMN) and salience network. In addition, serotonergic neurotransmission may regulate the symptoms of depression, including impulsivity, potentially by modulating the DMN. Kim and Kang investigated different reward systems implicated in IGD. For monetary reward, the IGD group exhibited stronger functional connectivity within the brain regions involved in motivational salience, whereas the group showed reduced functional connectivity the widely distributed brain areas involved in learning or attention. These differences in functional connectivity of reward networks, suggest that IGD is associated with an increased incentive salience or “wanting” process, which may serve as the neurobiological mechanisms underlying impaired goal-directed behavior.Kim et al. used the electrophysiological marker of late positive potential (LPP) to compare attentional bias in IGD and obsessive compulsive disorder (OCD). Increased LPPs in response to disorder-specific cues (game-related and OCD-related) were found in both IGD and OCD groups, respectively. These results indicate that LPP is a candidate neurophysiological marker for cue-related craving in IGD and OCD.Attentional bias toward addiction-related cues is also associated with incentive salience, but the pathophysiology of attentional bias in IGD is not well-understood, such as its relationship to compulsivity. Kim et al. investigated the neural correlates underlying the distorted self of individuals with IGD in relation to their satisfaction with basic psychological needs. Individuals with IGD had a negative ideal and actual self-image. Neurobiologically, dysfunction in the inferior parietal lobule associated with emotional regulation and negative self-evaluation was found in IGD. Recognizing that IGD often develops in adolescence, this self-concept problem should be noted and addressed with appropriate therapy approaches.Impairment of self-regulation is one of the major psychopathologies of addiction. Self-regulation ability is related to how well basic psychological needs are satisfied. These basic psychological needs, consisting of autonomy, competence, and relatedness, are important factors affecting individual growth and integration. Some individuals may rely on and overuse social media networks, as well as Internet games, in an attempt to meet basic psychological needs. Lee et al. identified IGD-associated miRNA markers by observing differentially expressed plasma miRNAs between the IGD and control groups. Through genome-wide screening of miRNA expression profiles and independent validation, three IGD-associated miRNAs  were discovered. Individuals with downregulation of all three miRNA are at high risk of IGD.Neurobehavioral phenotypes are epigenetically controlled by non-coding RNAs including microRNAs (miRNAs). Since miRNAs can be detected in blood (plasma or serum), circulating miRNAs have a definite advantage as non-invasive biomarkers in neuropsychiatric disorders. Hong et al. demonstrated that individuals with IGD were characterized by reductions in high-frequency heart rate variability while the subjects were playing their favorite online game. Their results suggest that an altered HRV response to specific gaming situations is related to addictive patterns of gaming and may reflect the diminished executive control of individuals with IGD while playing Internet games.Autonomic nerve system (ANS) dysfunction has also been associated with substance abuse and behavioral addiction. As the ANS responds to internal and external stimuli to maintain homeostasis, its function is closely related to adaptive adjustments in behavior strategies. ANS dysfunction likely contributes to the development and maintenance of loss of control over gaming, as individuals with IGD are unable to adjust their behavior strategies despite negative outcomes. ANS function can be assessed non-invasively by measuring heart rate variability (HRV). Chun et al. investigated altered brain connectivity associated with excessive smartphone use, and the relations between withdrawal symptoms, cortisol concentrations, and frontostriatal connectivity. They found that adolescents with excessive smartphone use had reduced functional connectivity in these regions related to cognitive control. Furthermore, Internet use withdrawal symptoms appear to elicit cortisol secretion, and this psychophysiological change may affect frontostriatal connectivity. These results provide important insights into the effects of excessive use of smartphones on brain functional connectivity in adolescence.As smartphone adoption and use has grown rapidly, there has been increased interest in the potential negative impact of excessive smartphone use. Seok and Sohn found that individuals with problematic hypersexual behavior have diminished executive control and impaired functionality in the right dorsolateral prefrontal cortex, which is a core feature shared across both addictive disorders and impulsive control disorders. In addition, Gola and Draps reported that CSB is related to increased ventral striatal reactivity during the anticipation of erotic stimuli, in support of the theory of incentive salience. They suggested that further studies should be undertaken to examine neurobiological differences in these two disorders.Gaming disorder and compulsive sexual behavior (CSB) disorder were recently included in the latest International Classification of Diseases (ICD-11). However, the WHO purposefully decided to classify compulsive sexual behavior disorder as an impulse control disorder, while gaming disorder was included to addictive disorders. Lee et al.'s study aimed to identify the neuropsychological factors that promote improved recovery from IGD. They reported that individuals with IGD who had not improved at 6-month follow-up were more likely to have higher aggression and harm avoidance at baseline, indicating that gaming problems among these more complex cases appear less likely to resolve spontaneously. The assessment of aggression and harm avoidance levels may help predict the course of IGD.This Research Topic also presents a series of novel studies that employ longitudinal designs, a design approach that historically has been quite limited in the IGD field. Park et al. investigated neural connectivity associated with treatment responses in patients with IGD using resting-state electroencephalography (EEG) coherence analyses. Compared with healthy controls (HCs), patients with IGD showed increased beta and gamma intrahemispheric coherence and increased delta intrahemispheric coherence of the right hemisphere at baseline. After 6 months of outpatient management including selective serotonin reuptake inhibitors, patients with IGD exhibited improvements in IGD symptoms compared with baseline, but they continued to show increased beta and gamma intrahemispheric coherence compared with that of HCs. These findings suggest that significantly greater intrahemispheric fast-frequency coherence may be an important neurophysiological trait marker of IGD.Kim et al. investigated the relative value of behavioral, temperamental, and physical factors in predicting risk/problematic Internet use (ARPIU) in adolescents. They found that, among boys, severity of Internet addiction correlated inversely with the 2D:4D digit ratio and novelty-seeking, and positively with reward dependence scores when controlling for depression scores. These relationships were not found in girls, suggesting the need for gender-sensitive approaches to prevent ARPIU in youth.The final category of studies in this Research Topic involved neurobiological diagnostic and treatment approaches. Kim and Hodgins proposes a transdiagnostic treatment model of addictions that targets underlying similarities between behavioral and substance use addictions. Their model highlights various component vulnerabilities, each with intervention possibilities, including: lack of motivation, urgency, maladaptive expectancies, deficits in self-control, deficits in social support, and compulsivity. In another paper relevant to this topic, Blum et al. introduced the “Precision Addiction Management” (PAM)™, the customization of neuronutrient supplementation based on the Genetic Addiction Risk Score test result, along with a behavioral intervention. Finally, Bae et al. examined bupropion as a treatment modality for IGD and gambling disorder. Bupropion showed promise for improving problematic behaviors in both IGD and GD, however there were differing pharmacodynamics across the two groups.The paper by In conclusion, the presented collection of original articles encompasses diverse research reports and review articles, with broad coverage of neurocognitive, neurophysiological, neurochemical, and neuroimaging research techniques. Together these articles demonstrate that the study of behavioral addictions from a neurobiological perspective is continuing to flourish and that there will be many exciting advances in this area that will improve our understanding, assessment, and treatment of individuals affected by these conditions.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Special properties of the polymeric nanomaterials (nanoscale size, large surface area to mass ratio and high reactivity individualize them in food packaging materials. They can be processed in precisely engineered materials with multifunctional and bioactive activity. This review offers a general view on polymeric nanocomposites and nanocoatings including classification, preparation methods, properties and short methodology of characterization, applications, selected types of them used in food packaging field and their antimicrobial, antioxidant, biological, biocatalyst and so forth, functions. Nanoscience and nanotechnology applications to the agriculture, food sector and food safety are relatively recent compared with their use in cosmetics and personal care products 60%), paints and coatings, catalysts and lubricants, security printing, drug delivery and pharmaceuticals, medical therapeutics and diagnostics, energy production, molecular computing and structural materials ,2,3,4. O0%, paintAccording to regulation (EC) = No 1935/2004 a good packaging should have the following functions: “to protecting the food from dirt or dust, oxygen, light, pathogenic microorganisms, moisture and a variety of other destructive or harmful substances. Packaging must also be safe under its intended conditions of use, inert, cheap to be produced, lightweight, easy to dispose or to reuse, able to withstand extreme conditions during processing or filling, impervious to a host of environmental storage and transport conditions and resistant to physical abuse” .Nanomaterials are applied in packaging and food safety are in various forms such as: polymer nanocomposites with high barrier properties, intelligent packaging, nanocoatings, surface biocides, active packaging, silver nanoparticles as antimicrobial agents, nutrition and nutraceuticals, nanosensors and assays for the detection of food relevant analytes  and bioplastics. (I)Improved packaging which offers improved mechanical properties as flexibility, enhanced barrier properties against water, gases, taint, durability, temperature/moisture stability, and so forth;(II)Active/bioactive food packaging offers antimicrobial, antioxidant or biocatalytic functions. It can be obtained by the incorporation of active/bioactive compounds into matrices used in existing packaging materials, or by the application of coatings with the mentioned functionality through physical or chemical surface modification. Active packaging is applied in food packaging, pharmaceuticals and consumer goods in order to improve shelf life, safety, or quality of packaged foods. Coating option is advantageous because the bulk properties of the packaging materials are preserved almost intact by using a minimum amount of active agent required to impart efficacy and therefore also cost is reduced.(III)Smart/intelligent packaging as a promising area for active packaging coating is developed by manufacture of nano(bio)sensors which can indicate quality of foodstuffs, of nano(bio)switch to release preservatives and nano-coatings as antimicrobial, antifungal, antioxidant, barrier coatings, external stimuli responsive materials and self-cleaning food contact surfaces. Intelligent inks such as nanoparticles and reactive nanolayers allow analyte recognition at nanoscale. Printed labels are applied to indicate: temperature, time, pathogen, freshness, humidity, integrity Carbon na of PVOH  are imprSilica nanoparticles (2nSiO) improve mechanical and/or barrier properties of PP [es of PP , mainly es of PP , of PVOHes of PP  or of stes of PP . Haloysite (HAL) represents a class of natural, non-toxic, non-swelling silicates, stable even at very high temperatures and show a good encapsulation and release capacity for bioactive compounds . They have a wide variety of applications including also as fillers and reinforcements for polymers. The HAL particles can adopt a variety of morphologies, as elongated tubules known as haloysite nanotubes (HNT), short tubular, spheroidal and platy particle shapes, the first one being the most common [Listeria monocytogenes, Clostridium perfringens and Staphylococcus aureus in skimmed milk agar being active and useful barrier to control food contamination [t common . HAL cant common . The HNTt common , by nanot common . Starch/mination . HNTs lomination . Graphene as atomically thin carbon sheets improve physical properties of host polymers at extremely small loading. Nanocomposites containing graphene are obtained by top-down strategies starting from graphite oxide, solvent- and melt-based strategies [rategies . Such narategies .Metallic nanoparticle incorporated into food contact polymeric matrices enhance mechanical and barrier properties, prevent the photodegradation of plastics and are effective antimicrobials  such as AgNP [ as AgNP . Copper, as AgNP .Lignin nanoparticles were tested for their antioxidant, antimicrobial activities and potential biological being effectively used as active agents towards different pathogen strains when is combined with PVA, chitosan CH, natural polymers and so forth [so forth ,113. Multifunctional nanocomposites contain usually a mixture of micro- and nanostructured materials such as essential oils, natural extracts and metal nanoparticles with antimicrobial/antioxidant agents/biological functions and they are also interesting for food contact materials.Increased use of the synthetic packaging materials  has led to serious ecological problems due to their non-biodegradability and waste accumulation. Biopolymers derived from natural resources as plants , animal  and microbial products, such as polyhydroxybutyrate have been extensively studied in the bio-based nanocomposites field  because Protein-based nanocomposites. In commercial applications, the following animal derived proteins used are: casein, whey protein, collagen, egg white and fish myofibrillar protein. The plant-based proteins also under large scale application consideration include soybean protein, zein (corn protein) and wheat gluten. Compared with non-ionic polysaccharide films, protein films, due to their more polar nature and more linear (non-ring) structure and lower free volume offer better oxygen barrier properties and lower water vapour permeability [2 or ZnO ware added to form nanocomposites with improved antimicrobial properties [2 nanoparticles significantly increased the tensile properties of whey protein film (from 1.69 to 2.38 MPa). Soy protein nanocomposite films exhibited reduced water vapour permeability, improved elastic modulus and tensile strength compared with neat polymer [eability . The traoperties ,121,122  polymer ,124. Zei polymer . It is l2 consumed during plant photosynthesis, water and humus through the activity of microorganisms [co-terephthalate) and thermoplastic starch which undergo degradation after various time period. Different biodegradation conditions can be considered: hydrolytic, composting, enzymatic, according to the final applications and the post-use of the new developed materials [Bionanocomposites (BNC) can be recycled/valorised or treated together with other organic wastes in composting facilities and produce compost, as valuable soil conditioners and fertilizers and so forth . They arrganisms . This beaterials .2, layered silicate and so forth) starch-based completely biodegradable materials with potential applications in food industry as edible films were prepared. [Combining the individual advantages of starch/synthetic or natural polymers and nanoparticles , while PLA/ organo-montmorillonite (OMMT) had the lowest biofilm formation because of the inhibitory effect of this NP during biodegradation test. Nanoclays influence the polymer bacterial degradation due to the affinity of the bacterium towards clay. The addition of nanoclays during composting process increases the PLA degradation rate due to the presence of hydroxyl groups from the silicate layers of these clays. As an example, due to the hydrophilic nature of nanocellulose, the cellulose nanocrystals (CNCs) increased the disintegrability rate of PLA.® RD, MMT showed a significantly higher mineralization of the films in comparison to the pristine PLA, mainly attributed to the reduction in the PLA lag time. PLGA/CNT films were degraded by hydrolytic degradation [PLA bio-nanocomposite films containing nanoclay with organo-modified , Halloysite nanotubes and Laponiteradation . CNTs inP. aeruginosa, systematically as the CNT loading increased from 0.1 to 10% w/w [MWCNTs reduce the biodegradation rate of PCL in the presence of  10% w/w .A coating is defined as a coherent layer formed from a single or multiple applications of a coating material to a substrate. Nanocoatings are ultra thin layers on the nanoscale <1–100 nm thick built-up onto surfaces ,133. NanSmart coatings” are coatings with multifunctional additional functions some also assuring thermal insulation, controlled release of active ingredients or self-healing functions. Nanocoatings can be built up with thickness of one molecular or multiple molecular layers. Nanocoatings are applied to various substrates such as metals, glass, ceramics, polymers and so forth  135,136136. Some®, nisin, pediocins), organic acids for food packaging or hygienic surfaces. Nanomaterial-containing coatings offer high performance materials and better processing properties than conventional coatings . Additives are added to a coating material in very small quantities. They can modify a large variety of properties, for instance, flow behaviour, surface tension, gloss, structure, UV and weather resistance. Depending on the desired function, nanotechnology-based functional coatings uses some nanomaterials as in the case of antimicrobial coatings containing silver, titanium dioxide, zinc oxide, some organic bioactive agents as polysaccharides, proteins, spice and herb extracts as essential and vegetable oils, bacteriocins  coated films and oxide evaporated films, for processed meat products  and so forth , offerinNano surface biocides  are obtained by incorporation such nanomaterials with antimicrobial properties onto packaging surface. Such food contact nano surface containing biocides maintain the hygienic conditions when applied to reusable food containers such as boxes and crates and inside liners of refrigerators and freezers. The most known coating technologies are immobilization using covalent (non-migratory) and non-covalent (migratory), layer-by-layer deposition, photografting chemistries and embedding for controlled release, schematically represented in In coating technology which uses the embedded agents for controlled release, the active agent is intended to migrate to the packaged good  while in non-migratory technologies, the active agent is intended to remain stable in the packaging matrix . Some techniques to prepare nanocoating are similar with those for nanocomposites obtaining, some are adapted and other are specific for this purpose. In some cases, the nanocoatings application required specialized equipment and complex procedures, such as, chemical vapour deposition or plasma spray techniques. The active agents are expected to migrate and exert their specific antimicrobial, antioxidant, biocatalytic, or nutraceutical functions within a packaged food. Active compounds are incorporated into polymeric materials by extrusion/blending and pip-coating techniques or solution casting.co-terephthalate) and cellulose derivatives, PVOH and so forth [Solution casting consists of dissolving the polymer in a suitable solvent and simultaneously incorporating the active compound of interest, followed by pouring this mix solution onto an inert surface. By the solvent evaporation, the films with desired functionality  result. It is mainly used at laboratory scale and can be translated at industrial scale by coupling with a wet-coating station and roll-to-roll system . Howeverso forth . Butnaruso forth . In commercial extrusion, the active compounds with polymeric materials in melted state, in a single or twin screw extruder by heat transfer where are incorporated a blend is obtained from which films can be formed. It also has serious limitations due to the intrinsic lack of thermal stability of many active compounds as it was already mentioned above, which can be lost either through degradation and evaporation. During the heat transfer involved in these unit operations the homogeneous distribution of active agent into matrix is difficult. The active compound release is controlled by packaging material characteristics such as the degree of affinity between it and the matrix, morphology and porosity and also by medium temperature. Multilaminated system in which the layer that harbours the active compound is covered by an adjacent layer could act as a barrier slowing release rate. In such case the bulk properties as tensile and thermal are changed therefore the coating technique is preferred. Sol-gel procedure is extensively used in obtaining of nano-based coatings. A viscous colloidal-sol is applied to a surface by dip, spray or spin coating processes onto surface. The thickness of the obtained layers varies from a few nanometres thick to 0.5 and 3 µm. The process occurs at low temperature, in a short time, is low energy consuming and more important without disruption of the structure or functionality of biocomplexes or organic aggregates, the nanocoating as a gel may exhibit a porous 3D network .There several types of sparaying guns as manual and automatic spray guns with air compressors, etc. Manual spray guns are available in various configurations to suit a wide range of applications and processes. HVLP (high volume low pressure) gun offers the following advatages: the spraying is controllable, high transfer efficiency, can be used for a wide variety of materials, it is inexpensive to maintain because needs a reduced preparation and clean-up, is portable, the material consumption is reduced by 40%.Their applications are: painting, food industry, pharmaceutical, dyestuff coating, oil, lubricating, rubber solution spray, textile spray, water based materials/paints, etc.They are already applied on a large scale both by individuals and industrially.coating. The main advantage of plasma treatments is that the modification is restricted to the uppermost layers of the substrate, thus not affecting the overall desirable bulk properties. To achieve approximately similar surface modification, gamma irradiation should apply only in mild controlled conditions [Surface immobilization of the bioactive compounds was performed for a wide range of inert hydrophobic packaging polymeric materials. Surface modification of polymers can be performed either by physical, chemical (wet) or biological methods. Physical methods are preferred over chemical techniques because they offer greater precision, ease of process control and environment friendliness. Classical physical methods for modifying polymer surfaces include flame and corona treatment, ultraviolet light, gamma-ray, ion-beam techniques, low-pressure plasma and laser treatment. Physical methods either modify the surface layer or an extraneous layer is deposited on the top of the existing material—nditions ,147. The physical methods are widely used due to their easier industrial scalability, no liquid reagents of any kind are needed in their application, thus is avoided the accumulation and handling of harmful waste . The maiCovalent attachment/conjugation of bioactive compounds to functionalized polymer surfaces is realized by: hydrophilic, bifunctional, and/or branched spacer molecules. The main advantage of covalent immobilization is high stability of the formed layer and the active compound does not migrate into the packaged food product to affect its quality and safety. Active agents can be covalently linked directly to the polymer surface or by use of a spacer or crosslinker, which either share a permanent covalent bond between the polymeric substrate and the bioactive compound, or promotes covalent bond formation between the activated substrate and the bioactive compound without forming part of that link (“zero-length” crosslinkers). It can also perform optimization of the surface functionalization by using a polyfunctional agent, increased number of functional groups, tethering a bioactive compound to a solid substrate via a spacer molecule, covalently attach a bioactive compound to the functionalized polymer surface via an intermediary. The immobilization technologies are potential versatile as once functional groups are introduced to the polymer surface, a range of bioactive agents  can be immobilized through standard chemical or bioconjugation techniques [chniques . Plasma and gamma rays assisted nanocoating and immobilization. Plasma treatment and gamma irradiation are energetic processing methods that create reactive spots to which either oxygen- or nitrogen containing groups can be implemented on surface and functional groups and radicals are created depending on the type of discharge gas/atmosphere used inside reactors. The energy levels of entities generated in cold plasma are comparable to the bond energies of organic compounds and thus can facilitate surface functionalization reactions. The extent of functionalization can be controlled by selecting the discharge parameters [2 and air plasma activation of inert surface of synthetic polymers [2 plasma activation [2) groups. In order to develop new multifunctional active polymeric surfaces with special properties for food packaging applications based on inert polymer such as polyolefins or polylactic acid and polyalkanoates, the following solutions have been applied:Activation of polymeric substrates by non-solvent, environment friendly methods by using of gamma-ionizing radiation or cold plasma gas discharge.Stable layers have been deposited onto activated polymeric substrates using different coupling agents for covalent linking of active/bioactive formulations. Selected bioactive compounds were: chitosan/chitin, lactoferrin, vitamin E, natural vegetable oils with high content of antioxidant compounds as phenols or flavonoids mixtures.rameters . On polypolymers  and amphtivation ,153, in Covalent grafting of bioactive compounds onto inert polymeric surfaces occurs by a two-step procedure:Polymeric surface activation.Step I. Surface activation techniques are applied in order to introduce the desired type and quantity of reactive functional groups which are able then to covalently attach a bioactive compound. The polymeric films/papers were exposed both to corona and to high-frequency plasma (air or nitrogen were used as discharge gas). The most important feature of irradiated polymers is the possibility of grafting for certain structures, because the radical sites are available for coupling. γ-exposure of polymeric films/papers was carried out in an irradiation machine at various irradiation doses from 2–30 kGy absorbed in air, at room temperature. Surface functionalization because of implementation of polar groups and radicals onto substrate surface provides desired changes in physical properties of the substrate surface  . AlthougCovalent bonding/grafting of bioactive compound onto activated polymeric surfaces. Step II. N-hydroxysuccinimide (NHS) or 1′-Carbonyldiimidazole (CDI) coupling agents were used.After the corona, plasma discharge or γ-irradiation pre-treatment, the polymer surface was enriched with oxygen-containing groups, such as carboxyl, carbonyl, hydroxyl, ester groups and/or nitrogen containing groups. The covalent attachment between a polymeric surface and an active compound is achieved by formation of amide, ether, ester and thioester bonds, created between the hydroxyl, amine, imine, carboxylic acid and thiol groups of the active compounds of interest which may possess intrinsically (or are incorporated in their structure) and the functional groups created on the substrate ,163,164.Comparing various methods of chitosan deposition onto cold plasma and γ-irradiation activated PE surface it was established that for the same concentration of the chitosan solution the most efficient method was the immersion in respect with homogeneity of surface and thickness of deposited layer but the electrospraying appeared to be more versatile method, because it allows a more precise control of the chitosan content deposited onto the surface by varying the deposition time. 2 or –OH groups of CHT with carboxyl group implemented on PE or PLA surface by radiation activation of surface. Other reactions were also possible because of the high reactivity of the active species created by plasma exposure/gamma irradiation and the functional groups of chitosan [2·day) while OTR of the uncoated PE is 3833.36 mL/(m2·day). The potentiometric and polyelectrolyte titrations, zeta potential determination showed that some amount of chitosan desorbed faster from the surface until equilibrium was reached and also that the grafted chitosan layer was more stable than the physically adsorbed one. In the case of grafting, a thin chitosan layer was irreversibly immobilized on the PE surface. The obtained materials combine the antibacterial, antifungal, bioadhesivity, biocompatibility, biodegradability of chitosan [Attenuated Total Reflectance Fourier TransformIinfrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and potentiometric titration proved CHT presence on the PE surface and also new bonds are evidenced mainly amide groups or ester groups because of interaction of –NHchitosan ,165 Chitchitosan ,167. witchitosan . The pH responsiveness was evidenced was evidenced by contact angle measurements using buffered solutions with pH varying in the 2–11 range and it appears by sudden switching from hydrophilic to hydrophobic surface at critical pH ≈ 6. The contact angle of the polyethylene surface remains constant over the entire studied pH range . Also, vegetable oils  and combinations chitosan/vegetable oils have been tested to combine antimicrobial activity of CHT with those of vegetable oils and their antioxidant, biological function and nutrition . They weLignocellulosic materials usually display a very low microbial resistance and microbial contaminations might be an additional issue to be taken into account. Conferring antibacterial activity to lignocellulose-based products may represent a main functional property which is useful not only for advanced food packaging  but alsoWet Methods involve both corrosive liquids to which the polymer substrates are directly exposed, like piranha solution (dissolved hydrogen peroxide and sulfuric acid), combined sodium hydroxide and sulfuric acid, chromic acid, potassium permanganate and nitric acid  and grafPhotografting method is a free radicals photografting procedure which occurs onto a polymer surface exposed to UV light in the 315–400 nm range in the presence of photoinitiators  and monomers which usually bear ketone groups . The actBiological methods consist in: pre-adsorption of proteins, drug, enzyme immobilization, cell seeding and pre-clotting . CVD, the substrate is exposed to one or more volatile precursors, which react and/or decompose on the substrate surface to produce the desired deposit [In typical  deposit . DiamondVTT Technical Research Centre of Finland developed a fully recyclable nanocoating for food and pharmaceuticals  which isBy layer-by-layer assembly deposition can prepare active packaging coatings by the incorporation of active agents either between layers or within the structure of an individual polyelectrolyte . Onto a Sono-Tek ultrasonic nozzles are suitable to depositing precision nanotechnology coatings for spray applications in research as well as production volume spray processes . Uniform® coating technique uses the plasma jets deposit micro-fine glass-like nano-coats which form a highly effective protective film on the packaging with minimum material usage. Plasma coats provide a perfect quality imprinting [Interior and possibly even exterior coating of the aluminium cans and tubes is required in the food industry because the direct contact between the product and the packaging can lead to aluminium corrosion and the food could spoil. For example, the pasteurization increases corrosive impact on packaging. PlasmaPlusprinting . Nobile printing  producedElectrospinning/electrospraying has recently gained much attention because of its versatility in processing a wide range of polymer and biopolymer materials. It has the ability to produce fibre/particle diameters within the submicron and nano range using electric field which cannot be achieved by using conventional-generating technologies. Nanofibers have wide applications in industry such as food packaging coatings, composite materials, carbon nano tubes, inorganic fibres and tissue scaffolds, filtration, medical, membranes and so forth . ElectroIn monoaxial electrospinning the device consists of a direct current high voltage supply, a metal plate collector sustaining the polymeric substrate, a pump and a syringe oriented perpendicularly to the metal plate with the low diameter needle  into a polymeric one so core-shell morphology is obtain which can increase the coating functionality and protection of the labile bioactive component and also the use of nanomaterials in other technology. Encapsulation of the vegetable oils in chitosan was achieved by coaxial electrospinning method and immediately a coating of the nanoparticles/nanofibres was deposited onto polymeric substrate [ubstrate ,189. ThiElectrospun fibres have a very high specific surface and porosity and they are able to encapsulate active substances. A wide range of applications are known for such fibres as improvement of physico-chemical and functional properties of biopolymer based materials by means of a controlled release of active compounds or by enhancing the dispersion of nano-additives into the biopolymer matrices. The nano encapsulation/ entrapment can reduce the amount of active ingredients needed to obtain certain functionality and also their controlled release ,188,190.Salmonella enterica below the detection limits at very low silver loading of 0.002 ± 0.0005 wt %. Also, polylactic acid (PLA)/Silver-NP/Vitamin E bionanocomposite has been prepared by electrospinning and they showed a good antimicrobial activity against both gram positive and gram-negative bacteria [A. Níger after 14 days, for the three multilayer samples coated with AgNP/polyethylene nanocomposites with the silver nanocomposite layer of AgNO3 suspension (black) all at 1.0 wt % of silver content depends on the coating method, the most efficient being spraying one which gave a reduction of more than 70% while laminated and casting method reduction were of 20% and 45%, respectively [An antimicrobial active multilayer system was obtained from a commercial polyhydroxyalkanoate substrate (PHA) and an electrospun PHA coating with in situ-stabilized silver nanoparticles (AgNPs) with satisfactory thermal, mechanical, barrier and antibacterial properties. It reduced the bacterial population of bacteria . As a rebacteria . The choectively .Nanocomposites and nanocoatings applications in food packaging depend both on the properties of matrices/substrates and activity of ingredients. Nanoclay composites  are used as flexible and rigid food packaging because of their excellent barrier characteristics in packaging of processed meats, cheese, confectionery, cereals and boil-in-the bag foods, items obtained by extrusion-coating process together with the paperboard are useful for fruit juice and dairy products and the beer and carbonated drinks bottles are manufactured by co-extrusion processes. For example, the PET bottles contain nanocore and nylon beer bottles with nanoclays show improved barrier characteristics. The active agents functions include antimicrobial, antifungal, antioxidant, biocatalyst, external stimuli responsiveness, additives that absorb undesirable component from food such as ethylene, moisture and odour and so forth They are incorporated into matrix or coating to enhance the quality and shelf life of the food and are released in a controllable manner, the procedure being much efficient then direct addition into the food because they may react with other food components and loss their activity or affect food sensorial characteristics.There are many formulations including such antimicrobials, antioxidants and biocatalysts, some of them being summarized in Hippophaë rhamnoides L.) leaves and inner bark of pine trees (Pinus silvestris) [Fusarium graminearum, Penicillium corylophilum and Aspergillus brasiliensis—and three potential pathogenic food bacteria as Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and also against Pseudomonas aeruginosa.Nanomaterials are more and more used to target bacteria in textile industry, marine transport, medicine and food packaging as antibacterial coatings and other materials . Antimicvestris)  used in vestris) . Such es2 and ZnO are non-toxic and approved by FDA as GRAS. TiO2 nanoparticles exhibiting bactericidal and fungicidal effect against Salmonella choleraesius, Vibrio parahaemoliticus, Staphylococus aureus, Diaporthe actinide and Penicilinum expansum. It was ultrasonically dispersed ethylene-vinyl alcohol copolymer films to obtain active packaging. It showed excellent bactericidal property under UV light [L. Monocytes and S. Enteritis.TiOUV light . BecauseUV light . ZnO is + ions by AgNPs dissolution which then bind to electron donor groups in biological molecules containing sulphur, oxygen or nitrogen [E. Coli, Enterococcus faecalis, Staphylococcus aureus and Epidermidis, Vibrio cholera, Pseudomonas aeruginosa and putida and fluorescens and oleovorans, Shigella flexneri, Bacillus anthracis and subtilisi and Cereus, Proteus mirabilis, Salmonella enterica typhmurium, Micrococcus luteus, Listeria monocytogenes and Klebsiella pneumoniae [Have been proposed several mechanisms for the antimicrobial activity of nanoparticles as: directly interactions with microbial cells and interruption of the trans-membrane electron transfer, disrupting/penetrating the cell envelope, oxidizing cell components or producing secondary products as reactive oxygen species (ROS) or dissolved heavy metal ions. Secondary processes also can occur. For example, the antibacterial activity of silver ion is reduced by the protein rich food because it can bind the cysteine, methionine, lysine and arginine . Nanosilnitrogen . Silver nitrogen . It was eumoniae ,263 someeumoniae  in diffe® technology are based on silver-functionalized nanoclay. MgO and ZnO nanoparticles could provide cheap, safe alternative to expensive nano-sized silver as a heavy metal not suitable for human contact. The combinations of α−tocopherol with nisin or chitosan as coating or biocomposites confer both antimicrobial and antioxidative properties [The NanoBioMatters highly efficient antimicrobial product and BactiBlockoperties ,140,225.Antimicrobial activity of the chitosan and engineered nanoparticles is explained by several mechanisms including interactions between positively charged chitosan and negatively charged cell membranes, increased membrane permeability and rupture and leakage of intracellular material. They are ineffective at pH values above 6 because of the absence of protonated amino groups . AnotherCarbon nanotubes also have antibacterial properties. They are efficient against E. coli, possibly because puncture microbial cells [al cells  but is pOxidative degradation of food products happens during transport and storage producing lipid rancidity, colour loss  and vitamin degradation. The antioxidants  are commonly incorporated in food packaging by blending with polymers during processing  ,268. TheEnzymes onto solid support materials provide biocatalytic coatings for active packaging by catalysing some reactions. Biocatalysts are used in ingredients production and breakdown of undesirable components which are harmful or may decrease product quality. Biocatalysts are very sensitive, therefore the immobilization method onto and into solid supports must preserve their thermostability, optima pH and solvent stability and usually so-called “in-package processing” is applied to avoid changes during processing, transport and storage. Antimicrobial enzymes  are incorporated into active packaging coatings via blending, non-covalent binding for controlled release and covalent immobilization ,271,272.A critical issue in food packaging is that of migration and permeability ,274 to aN-isopropylacrylamide)-based microgels and assemblies have a diverse range of applications for example, biosensing, smart coatings and so forth [Stimuli responsive nanocomposites/nanocoatings prepared using “stimuli-responsive, “smart,” “intelligent” or “environmentally sensitive” polymers ,284,285.so forth . Sensory Packaging is a category of devices used in intelligent packaging as nanosensors to monitor and report on the condition of food, communicate the degradation of product or microbial contamination and give information on history of storage, avoiding inaccurate expiration date increasing security and food safety. It helps food industry and retailers to now if the food package has been opened or tampered. This could be achieved by using a nanocrystalline indicator in form of an oxygen intelligence ink printable on most surfaces. This ink contains an UV light activated nanocrystalline particles of a semiconductor  or nanophosphourus particles. These particles are white in daylight but will fluoresce when exposed to light of certain wavelengths. The colour pigments can be also used as micro-colour codes in packaging and labelling applications. The development of sensory packaging which monitor the conditions of pharmaceuticals and foods are those affected by changes in temperature, humidity and shock. Nanobarcodes developed by Nanoplex are written into fluorescent microspheres by photobleaching. Coating on PET bottles will expand the packaging of sensitive juice or beer beverages 2 into lipid matrix results in rancidity) and in some cases nanoparticles of bioactive compounds are intended to be released deliberately.Consumers are hesitant to buy nanotechnology foods or food with nanotechnology packaging. However, some results suggest that nanotechnology packaging is more beneficial than nanotechnology foods. The main concern with the application of the nanotechnology in food packaging is related to the fact that the very small sizes of the nanoparticles have different chemical and physical properties in respect of macroscale materials and it is possible they could cause health problems. New packaging materials must have good barrier properties to oxygen, carbon dioxide, water vapour and flavour compounds. This controlled release packaging is another example of a nanomaterial application in active packaging. Nanoclays can be used as carriers for the active agents with high efficacy because it is highly dispersed in the polymeric matrix and, hence, exposed more efficiently to the substance on which it is required to act. Potential implications for consumer safety are migration of nanoparticles. They can enter into body through ingestion, inhalation or dermal contact leading to health effects of exposure to some insoluble, persistent nanoparticles. Such health effects are currently not known. Nanoparticles also migrate to foodstuffs with possible adverse effects on food quality. Other concern is with degradability of bio-polymers and formation of degradation products with possible adverse effects. Also, may appear potential environmental impacts of nano-polymer composites and some problems with end-of-life treatments as recycling, re-use and disposal. Detecting the migration of nanomaterials requires more sensitive analytical techniques due to the complexity of the nanomaterials and because they represent only a very small portion of the bulk food. Migration into food can cause both undesirable organoleptic changes , starting silver concentration, temperature, time and choice of contact medium are all factors which may have an effect on the extent of silver ion migration into food simulants. In general, the rate of migration increases when nanosilver is coated onto the food packaging material or surfactants are added, when the storage temperature and length of storage increases and the acidity of the contact medium increases. There appears to be a specific time of storage, after which a steady state release of silver is achieved. This is supported also by a repeat contact migration experiments, which found silver migration decreased considerably (by an order of magnitude) after first contact. Nevertheless, there is some evidence to suggest that if silver nanoparticles do migrate into food/food simulants, they would most likely dissolve quickly into ionic silver. The majority of the migration studies found for nanosilver food packaging composites have shown levels of migration of ionic silver into foods and food simulants below the European specific migration limit (SML) of 0.05 mg Ag/kg food, suggesting low consumer exposure and subsequently low risk of adverse effects. However, there are also several studies, in which migration exceeded this limit. This indicates that for new food packaging products containing nanosilver, the migration experiments should be conducted in each particular case. Migration of intact nanoparticles into food simulants is negligible, implying consumer exposure to these materials is likely low. This suggests there is low potential for safety issues related to the nanosize level of the materials incorporated into food packaging. If they migrate in nanoparticulate form, it would be anticipated at the resulting low concentration in food that many of the metal oxide nanoparticulates would likely dissolve into their ionic forms upon contact with acid foods or stomach acid. Theoretically, potential consumer exposure to nanomaterials incorporated into food packaging will appear if: they migrate into foodstuffs or drinks from the packaging, or if the nanocomposite polymers degrade and ‘dissolve’ into food or drinks. Migration can theoretically occur if nanoparticles desorb from the surface of the packaging material due to weak bonding at the surface , diffuse into foods as a result of a concentration gradient, or dissolve resulting in ions released into food ,290. HowThe companies producing nanoreinforced food packaging materials are Color Matrix Corporation manufacturing Imperm a high barrier nylon multilayer bottles films; Mitsubishi Gas Chemical Comp. Nanocore producing NANO-N-MXD6 for PET bottles; Lanxess for Durethan KU2-2601; Honeywell polymer with Aegis NC and OX as coating PA6 on paperboard and coinjection PET bottles , NanoSea® as multilayer PET bottle and sheets is used to improve barrier properties and Nylon MXD6 Nylon MXD6-Ultra Barrier System [® KU 2-2601  nylon nanocomposites for films and paper coating designed for medium barrier applications, requiring excellent clarity; Aegis®  a polymerized nanocomposite film incorporating an active oxygen scavengers and passive nanocomposite clay particles.Nanocor Inc. Deveoped Impermr System , Duretha−2 g/(m2 day), transparency 86% (PET 87%) at 550 nm, stretchability > 5%, average roughness 10.3 nm. Sidel trade name used for less sensitive foods are a coating of about 200 nm thick obtained inside PET bottle wall by acetylene plasma. “Micro-pore-plugging” film as single nanolayer on PET, manufactured by Tera-Barrier, provides transparency and stretchability for applications in food, medical and other packaging markets. Coating of 400 nm is obtained by slot die and has WVTR (40 °C and 100% RH) of 5 × 10Barrier films are manufactured by Clairiant and Nanocore from PP and respectively Nylon with organoclay for packaging or PET beer bottle. Purdue University developed a large-scale manufacturing process that may change the way some grocery store foods are packaged. This new manufacturing process uses cellulose nanocrystals as advanced barrier coatings for food packaging. The Purdue manufacturing technique is a roll-to-roll manufacturing process using waterborne polymer systems being scalable . By theiThe global trade of active food packaging was estimated to be around US $12,000,000,000.00 in 2017 . The actNanotechnology has the potential to generate new food products and new food packaging. Analyses of individual data showed that the importance of naturalness in food products and trust were significant factors influencing the perceived risk and the perceived benefit of nanotechnology foods and nanotechnology food packaging ,302,303.Nanotechnology has been found to be a promising technology for the food packaging industry. It has proven capabilities that are valuable in packaging foods, including improved barriers; mechanical, thermal and biodegradable properties; and applications in active and intelligent food packaging including anti-microbial agents and nanosensors, respectively. However, the use of nanocomposites in food packaging might be challenging due to the reduced particle size of nanomaterials and the fact that the chemical and physical characteristics of such tiny materials may be quite different from those of their macro-scale counterparts. Migration studies must be conducted to determine the amounts of nanomaterials released into the food matrices . Nanosca


Candida parapsilosis after TEA in a 57 year-old Caucasian woman with a history of hypertension, depression, and three previous surgical procedures to the affected limb. A fungal PJI by the organism C. parapsilosis following TEA has not been previously reported. Successful eradication of the fungal infection was achieved utilizing resection arthroplasty; placement of an amphotericin, vancomycin, and tobramycin-impregnated cement spacer; and 6 months of organism-specific antifungal medication. Although the patient was clinically ready for reimplantation, she passed away due to unrelated issues before reimplantation could be performed. While PJI is a devastating complication following TEA, a fungal infection is a rare complication that imposes difficult challenges to the treating surgeon. With our case report, we hope to contribute to the overall knowledge of fungal infections associated with TEA and describe our successful treatment of this complex case.A specific treatment protocol for managing fungal infections after total elbow arthroplasty (TEA) does not currently exist. The purpose of this report is to describe our experience and outline our treatment algorithm for a rare case of prosthetic joint infection (PJI) following a TEA. We present a case of a PJI due to  Advances in surgical technique along with innovations in implant design have allowed total elbow arthroplasty (TEA) to become an increasingly common and effective treatment option for patients with rheumatoid arthritis, persistent joint instability, unsalvageable distal humerus fractures, and end-stage osteoarthritis –5. WhileCandida parapsilosis following a TEA in a 57 year-old Caucasian woman. To our knowledge, only one other case of fungal PJI after TEA has been reported in the orthopaedic literature; however, a case of fungal PJI by the organism C. parapsilosis has not been previously reported.The rate of prosthetic joint infection (PJI) in TEA has been reported to be 5% to 8%, a rate higher to that of both total knee and total hip arthroplasty –3. The mTo date, a specific treatment protocol for managing fungal infections following TEA does not exist. The purpose of this report is to describe our experience and outline our treatment algorithm for this rare cause of PJI following a TEA.A 57 year-old, right hand-dominant female sustained a right distal humerus fracture from a fall in mid-2009; her past medical history was significant for hypertension and depression. The patient underwent fracture fixation and collateral ligament repair at an outside institution. Due to persistent elbow pain and instability, revision collateral ligament repair was performed in late 2009. In early 2010, the patient underwent lateral collateral ligament reconstruction utilizing allograft tissue by her initial surgeon due to reported recurrent elbow instability.Candida parapsilosis, and she was subsequently placed on chronic fluconazole , chronic corticosteroid use, and chronic antibiotic use , 7, 8. SAspergillus terreus. Initially, the patient required a resection arthroplasty due to a persistent draining sinus and a coagulase-negative staphylococcus infection. After staged treatment with a vancomycin-impregnated cement spacer and 6 weeks of ciprofloxacin and vancomycin for a simultaneous Enterobacter cloacae infection, the patient underwent subsequent TEA reimplantation but developed clinical signs of infection at six months postoperatively. The joint aspiration grew Aspergillus terreus, which was treated with 8 weeks of intravenous voriconazole and vancomycin. Unfortunately, while receiving immunomodulators for a rheumatoid flare, symptoms recurred requiring a repeat resection arthroplasty and course of antifungal medications. For patients such as this with immune-mediated disease undergoing TEA, limitations exist on the interpretation of inflammatory markers such as ESR and CRP. Such patients can have elevated basal levels of ESR or CRP that have the potential to cause false-positive tests, and the use of immunosuppressive therapies such as disease-modifying antirheumatic drugs (DMARDs) can also affect the accuracy of test results [To the best of our knowledge, only one other case of fungal infection following a TEA exists in the literature. Kwong et al.  reported results .Due to the rarity of fungal infection in TEA, specific treatment guidelines for this pathology do not exist. Currently, the Infectious Disease Society of America (IDSA) recommends against sole treatment with systemic antifungal agents or one-stage exchange arthroplasty in fungal PJI in TKA and THA . InsteadC. parapsilosis fungal PJI after TEA utilizing resection arthroplasty, placement of an amphotericin, vancomycin, and tobramycin-impregnated cement spacer and 6 months of organism-specific antifungal medication. With our case report, we hope to contribute to the overall knowledge of fungal infections associated with TEA and describe our successful treatment of this complex case.While a PJI remains a devastating complication following TEA, a fungal PJI imposes additional challenges to the treating surgeon. Patients with multiple medical comorbidities, an immunocompromised state, and a history of multiple elbow procedures may be at an increased risk of developing a fungal infection. Treatment guidelines for the management of fungal infection after TEA currently do not exist. Here, we describe the successful treatment of a


Bone regeneration represents a complex process, of which basic biologic principles have been evolutionarily conserved over a broad range of different species. Bone represents one of few tissues that can heal without forming a fibrous scar and, as such, resembles a unique form of tissue regeneration. Despite a tremendous improvement in surgical techniques in the past decades, impaired bone regeneration including non-unions still affect a significant number of patients with fractures. As impaired bone regeneration is associated with high socio-economic implications, it is an essential clinical need to gain a full understanding of the pathophysiology and identify novel treatment approaches. This review focuses on the clinical implications of impaired bone regeneration, including currently available treatment options. Moreover, recent advances in the understanding of fracture healing are discussed, which have resulted in the identification and development of novel therapeutic approaches for affected patients. In the majority of patients with a long bone fracture, the fracture ends meet to reform a mature and mechanically stable bone: Especially in the case of long bone fractures, this is usually achieved by surgical state-of-the-art treatment. In a significant number of cases, however, fractures do not heal and show no or insufficient signs of union . The devThe reasons for the formation of non-unions are complex and manifold . The preEven though stiff and sturdy, bone tissue is biologically very active and constantly remodeled through the balanced activities of bone-resorbing osteoclasts and bone-forming osteoblasts. Similar to bone remodeling in healthy skeletal tissue, bone regeneration represents a complex process which basic biologic principles have been evolutionary conserved over a broad range of different species. It is known that bone represents one of the few tissues that can heal without forming a fibrous scar. As such, the process of fracture healing is considered to recapitulate bone development and resembles a unique form of tissue regeneration. In this context, callus remodeling represents an important aspect of fracture repair, resulting in bone quality and strength comparable to the pre-fracture state .Fracture healing is traditionally subdivided into various biological phases which follow a chronological order. These particular phases help to understand the biology of fracture repair on a mechanistic level, however they cannot be strictly separated as they occur simultaneously at different sites and at different rates throughout the fracture callus. In general, tissue healing is characterized by an inflammatory, a proliferation/differentiation, as well as a remodeling phase. The same applies for fracture healing but has to be extended by matrix ossification, which occurs between the proliferation/differentiation and the remodeling phase .As a first consequence of fracture and vascular disruption, bone marrow and vascular leakage create a local hematoma containing bone and immune cells . The forPro-inflammatory cytokines in the innate response to a fracture include interleukin (IL) -1β, IL-6 and tumor necrosis factor-α (TNF-α) . While IWhile the above described inflammatory processes subsequently decline, the reparative phase begins in parallel by the formation of granulation tissue. As mesenchymal stem cells emerge and differentiate into fibroblasts, they produce extracellular matrix proteins such as collagens type I and II ,24. Due On a molecular level, these reparative processes are mediated by particular growth factors like transforming growth factor-β (TGF-β), bone morphogenic proteins (BMP), fibroblast growth factor (FGF), insulin like growth factor (IGF) and platelet derived growth factor (PDGF). TGF-β acts as pleiotropic growth factor stimulating undifferentiated MSC proliferation while BMPs promote MSC differentiation to chondrocytes and osteoblasts. FGF and PDGF act mitogenic on MSCs and osteoblasts. While FGF is critical for angiogenesis, PDGF also acts in macrophage chemotaxis. IGF promotes proliferation and differentiation of osteoprogenitor cells .Since the soft callus itself only provides basic mechanical stability, it is subject to ossification that is achieved through incorporation of calcium phosphate into the extracellular matrix. Ossification is distinguished in intramembranous and enchondral ossification which occur under different conditions and are likely to occur simultaneously at different zones in the fracture site . In intrIndependent of whether bone is restored via intramembranous or enchondral ossification, mechanically inferior woven bone is created at first. Woven bone is characterized by a random organization of collagen fibers and mineralized tissue. In order to generate mechanically stable lamellar bone, the woven bone is subsequently remodeled to form mechanically competent lamellar bone. Similar to healthy bone of which 10% is replaced and remodeled each year in humans , the traThe outcome of bone regeneration depends on both the biological and the mechanical environment. The mechanical environment primarily influences whether bone heals primary (direct) or secondary (indirect). Primary bone healing can be observed in anatomically reduced fractures with rigid fixation and interfragmentary compression. In this case, the fracture heals only through bone remodeling, without the formation of a fracture callus ,28. In cAs both, intramembranous and enchondral ossification, are observed in secondary bone healing, the degree of fixation determines the principle way of ossification. Experimentally, Thompson et al. could show that externally stabilized fractures healed primarily via intramembranous ossification in a standardized mouse model of tibial fracture, while the expression of collagen type IIa pointed towards enchondral ossification in non-stabilized fractures. Moreover, the authors concluded that the mechanical environment influences cell fate and thus the course of ossification as soon as day 4 in their mouse model, highlighting the importance of the early fracture hematoma for successful bone regeneration .Regardless of past, current or future treatment concepts, a detailed medical history of the patient and the individual case is paramount for an optimal and individual therapy of non-unions. For this purpose, it is important to establish a chronological list of the previous course of treatment with all therapeutic interventions, as well as risk factors, pre-existing illnesses and long-term medication. Often neglected is the thorough study of medical and surgical reports including microbiological and histological findings, the previous drug history and the chronological course of the infection parameters.Clinical symptoms of non-union include persistent, often relapsing stress-related pain in the area of the former fracture. Malposition or load-dependent and reproducible instability may be present. Clinical examination should include a detailed inspection of the soft tissue, the scars and a blood flow assessment at the affected limb. In case of evidence for impaired vascularization, CT-angiography is indicated. Moreover, a detailed study of existing radiographic imaging should be performed as it is crucial not only to review the latest radiographs, but also the initial accident images, the intraoperative fluoroscopy and the follow-up images during the healing process chronologically. Only in combination of all relevant findings it is possible to classify a non-union as precisely as possible and to establish an individual treatment strategy. With correlating clinical symptoms, a whole-limb radiograph should be performed in order to detect malposition or axial deviation. For accurate analysis and pre-operative planning, a computed tomography is of utmost importance. In selected cases, further imaging using contrast-enhanced CT, magnetic resonance imaging, angiography or PET-CT provide further information regarding osteomyelitis, blood perfusion, and soft tissue coverage.Restitutio ad integrum is the intended goal of any non-union treatment. In addition to achieving a pain-free and load-stable situation, the chosen approach ideally results in full restoration of the axis and length of the affected limb, as well as in complete healing of the bone defect. Apart from various surgical approaches, conservative treatment options are also available in individual cases, e.g., in delayed unions with prior sufficient fracture immobilization through stable osteosynthesis. In the case of non-unions with no signs of osseous consolidation, however, revision surgery is usually indicated. The treatment is usually based on the “Diamond Concept” [Concept” , a conceConcept” . Based oConservative treatment of delayed and non-unions requires sufficient mechanical stability as well as an intact biological environment at and around the fracture site. Therefore, conservative therapeutic approaches are mainly considered in early phases of non-union treatment. In addition to stimulate fracture healing through an increased mechanical strain , low-intensity pulsed ultrasound (LIPUS) or extracorporal shock wave therapy (ESWT) may be applied locally ,34. DepeWhile the efficacy of LIPUS and ESWT require further clinical research, to date the standard treatment of fracture non-union is surgical. The surgical intervention begins with the careful exposure of the fracture site and the debridement of sclerotic edges in order to obtain a vital and bleeding surface. Thereafter, the intramedullary cavities of fragments may be opened in order to facilitate the blood flow. As vital fracture ends are aligned rigid fixation may be performed. In many cases, resection of the malunion consisting of fibrous, often atrophic tissue, results in bone defects, which require bone grafting to bridge the fracture ends and to facilitate bone healing.3 of bone marrow aspirate and has gained wide acceptance in the treatment of non-unions over the last years [Despite all efforts in the research of artificial bone substitutes, the harvest and transplantation of autogenous bone from the iliac crest still represents the “gold standard” to support bone healing in non-unions as it combines osteogenic, osteoinductive and osteoconductive properties. However, autografts are only limited available and accompanied with high morbidity during harvest, including wound infection and postoperative pain ,37. An ist years ,39. Studst years ,41. Evenst years .Allogenic bone grafts provide a relatively safe alternative as autografts have limited availability and harvesting is associated with longer operation time and donor site morbidity. Allografts are usually used as cancellous bone chips providing some degree of structural strength. Due to their porous nature, ready-to-use bone allografts have very good osteoconductive properties. However, processing in terms of sterilization and storage causes loss of osteogenic and osteoinductive capability . While t3), or if infection is suspected, a multi-stage procedure with temporary implantation of a cement spacer to induce a Masquelet membrane is recommended . If the bone defect is critical  is a polypeptide hormone composed of 84 amino acids and is produced by the parathyroid glands. PTH regulates calcium hemostasis by increasing calcium resorption from bone and kidney directly as well as indirectly via vitamin D from the intestines . In 1997Intermittent PTH enhances bone formation by inhibiting proliferation of osteoblast progenitors determining them for osteoblast differentiation and inhibiting osteoblast apoptosis . The posBukata et al. addressed the use of teriparatide in cases of impaired fracture healing. 88% of the patients in their study either failed primary surgery, presented with delayed or non-union or were high-risk patients for impaired healing. Of 145 patients with complicated fracture healing, a 93% success rate with 12 weeks of adjuvant teriparatide treatment at 20 µg daily was observed. The authors propose teriparatide as a potential agent in complicated fractures and point out the need for prospective clinical trials . FurtherAs of now, most studies on the use of teriparatide in fractures focused on the cancellous metaphyseal region. In human osteoporosis, Jiang et al. could show a positive effect of teriparatide on cancellous bone architecture as well as cortical thickness after 12-month of treatment . Ogura eWnt signaling is crucial in regulating diverse processes in tissue development and also plays a pivotal role in bone formation. Mechanistically, Wnt ligands bind to the Frizzled receptor (Fz) which forms a complex with the LDL receptor-related protein 5/6 (LRP5/6) co-receptor. Subsequently, LRP5/6 and Fz deactivate the β-catenin destruction complex, which leads to accumulation of β-catenin within the cell. β-catenin translocates to the nucleus, where it regulates the transcription of specific Wnt target genes and thus osteogenesis . For exaAs mentioned above, Wnt ligands induce various transcription factors. Runx2 is considered the master transcription factor in osteoblast differentiation, as it controls the osteoblast-specific cis acting element present in the promoter of several osteoblast specific genes and thus regulates the expression of major bone matrix proteins . GeneticSclerostin, a product of the SOST gene, is expressed in osteocytes and a well-established inhibitor of Wnt signaling and thus, bone formation  Figure . SclerosTo achieve enhanced osteogenesis, osteoconductive scaffolds can be used in combination with osteoinductive agents. Such agents were first described in the 1960s, when Urist showed that acellular, devitalized and decalcified bone matrix could induce bone formation in ectopic tissue . Today, Recombinant human BMP-2 (rhBMP-2) and rhBMP-7 were approved by the FDA for specific clinical indications. The use BMP-2 was shown beneficial in the treatment of acute tibial fractures . AugmentIndependent of their potential adverse effects, BMPs are still considered potent inductors of bone formation and researchers are trying to adapt these proteins in order to make their use save. Since efficacy of BMP-2 was shown to be dose dependent, the described side effects may, at least partly, be explained by relatively high concentrations used . The rigAs the strive for iliac crest autograft substitutes continues, PDGF was found to be a promising candidate to promote bone regeneration. PDGF is found in the early fracture hematoma where it is expressed by endothelial and mesenchymal cells following the primary insult. In the course of fracture healing, it is also expressed by osteoblasts, chondrocytes and even osteoclasts . PDGF acIn terms of fracture healing PDGF must be used with caution. Imatinib, a monoclonal antibody inhibiting tyrosine kinase activity, was shown to promote osteoblast differentiation by inhibiting PDGF receptor β (PDGFRβ) signaling . FurtherPreclinical trials on the use of PDGF overexpressing hematopoietic stem cells in mice demonstrated increased trabecular bone formation and trabecular connectivity as well as decreased cortical porosity, adding up to a 45% increase in bone strength with low dose treatment . Those eFGFs describe a large family of 22 ligands binding to four receptor tyrosine kinases essential for proper tissue development and metabolism . The impFGF-2 interacts closely with other mediators involved in bone metabolism described above. Xiao et al. described a low molecular weight isoform of FGF-2 to carry out the anabolic FGF-2 function by modulating Wnt signaling . OverexpMost research has been focused on enhancing the anabolic effects of FGFs, while blockage of inhibitory effects of FGFs could be just as valuable. Overexpression of FGF-23 has been shown to inhibit osteoblast differentiation and matrix mineralization in vitro . For ostBesides FGFs itself, FGF receptors (FGFR) and their downstream signaling have moved into focus of research. Impaired FGFR3 signaling was found to be associated with defects in skeletogenesis. While activating mutations cause small statue, inactivating mutations result in tall statue ,128. In As described above, research in the past decades has identified various osteoanabolic agents which are currently under further investigation and some of which are already applied clinically. While systemic diseases such as osteoporosis increase the risk for impaired bone regeneration, young and healthy individuals may also experience non-unions, pointing towards a local pathology. From a clinical point of view, local therapy is therefore preferable as it additionally minimizes the risk for possible side effects. The locally applied agent’s potential to promote bone formation will, however, depend on a suitable scaffold, allowing adequate cell migration, proliferation, and differentiation as well as matrix production and degradation. While currently used natural bone grafts have been discussed above, an increasing number of artificial bone grafts may be applied in the treatment of fracture non-union. Importantly, artificial bone grafts, as an alternative to natural auto- and allografts, are not associated with donor site morbidity or the possibility of immunogenic complications. Additionally, they exist, at least theoretically, at unlimited availability. The ideal bone graft shows osteogenic, osteoinductive and osteoconductive properties, is perfectly biocompatible with no immunogenic reaction, biodegradable without harmful byproducts, allows or even induces neovascularization, and provides a certain degree of mechanical stability.Calcium based bone grafts include calcium phosphate ceramics, calcium phosphate cements and calcium sulfate. Calcium phosphate-based biomaterials were shown to have osteoconductive properties that are attributed to their microporous structure . CalciumCalcium phosphates such as hydroxyapatite (HA) and TCP have been widely investigated and used in bone defects. In healthy bone, HA is produced by osteoblasts and represents the major anorganic component of the bone matrix. The biocompatibility of HA bone grafts is thought to result from its structural similarity to natural bone matrix . ScaffolNowadays however, modern scaffolds are generally produced from a biodegradable polymer and the ceramic component. Advanced biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA) in the combination with a ceramic were able to overcome limitations of conventional ceramic bone substitutes ,144. AddBioactive glass describes silicate-based ceramics with bioactive properties. It was first described in 1971 and recognized for its ability to bind host bone . BesidesOrganic bone grafts are nanocomposites of mainly type I collagen mimicking the natural collagen scaffold. Mineralization is mainly attributed to non-collagenous proteins (NCPs) and collagen is considered to provide a passive mechanical support for mineralization . HoweverAs new technologies are developed and, in several cases, have revolutionized whole industries, the field of medicine is no exception. Huge progress has been made in the field of tissue engineering. One the one hand, advances in biology allow the production of bioinspired tissue which resembles the properties of native tissue. On the other hand, modern technologies such as 3D printing enable scientists to produce customized implants with high structural complexity on the nanoscopic level. In the case of non-unions with impaired local biology combined with the challenging tissue characteristics of bone and the often-large defects, patients could profit tremendously from these novel approaches, and current expectations are high. Up to now, 3D printed scaffolds are used in orthopedic trauma surgery to improve pre-operative planning with personalized anatomic models, and to overcome complex anatomic differences or defects which cannot be met with conventional implants . 3D prinDespite these limitations, recent advances in biomaterials are promising. Bioinspired composite matrices composed of a crystalline, rod-shaped nanoHA core and an amorphous silica sheath (Si-nHA) were shown to have good biocompatibility, osteogenic differentiation, vascularization and bone regeneration potential. In a critical size femoral defect rat model, this matrix resulted in complete bridging and union of the bone gap . Turner Since tissue, including bone repair, is a precisely regulated process with a wide diversity of growth factors and cell types exerting their function at a different time, sequential stimulation is attracting attention. Incorporation of growth factors and even cells into implants and their release at a desired point of time promises a more specific and hopefully effective support in tissue regeneration. Electrochemical-, pH- and UV triggered agent release were demonstrated ,169,170.One last aspect of implants is that of degradability. Patients with implant-associated pain or foreign body feeling may require an additional surgical intervention for implant removal, with the potential for peri-operative complications . TherefoDespite tremendous scientific and clinical effort, impaired bone healing still represents a complex and challenging complication following a fracture. A detailed case history, state-of-the-art diagnostics, and individualized treatment concepts are crucial for optimal patient outcome. Scientific advances in deeper understanding the molecular processes governing fracture healing have resulted in the identification of key mediators which can potentially be targeted to promote bone regeneration. The improvement of currently available bone substitute materials and the development of innovative biomaterials have significantly contributed to expand available treatment options. Therefore, further research and identification of novel therapeutic approaches with adequate safety profiles fulfills an essential clinical need, to promote bone regeneration and restore bone defects in patients suffering from non-unions.


Bone fractures and segmental bone defects are a significant source of patient morbidity and place a staggering economic burden on the healthcare system. The annual cost of treating bone defects in the US has been estimated to be $5 billion, while enormous costs are spent on bone grafts for bone injuries, tumors, and other pathologies associated with defective fracture healing. Autologous bone grafts represent the gold standard for the treatment of bone defects. However, they are associated with variable clinical outcomes, postsurgical morbidity, especially at the donor site, and increased surgical costs. In an effort to circumvent these limitations, tissue engineering and cell-based therapies have been proposed as alternatives to induce and promote bone repair. This review focuses on the recent advances in bone tissue engineering (BTE), specifically looking at its role in treating delayed fracture healing (non-unions) and the resulting segmental bone defects. Herein we discuss: (1) the processes of endochondral and intramembranous bone formation; (2) the role of stem cells, looking specifically at mesenchymal (MSC), embryonic (ESC), and induced pluripotent (iPSC) stem cells as viable building blocks to engineer bone implants; (3) the biomaterials used to direct tissue growth, with a focus on ceramic, biodegradable polymers, and composite materials; (4) the growth factors and molecular signals used to induce differentiation of stem cells into the osteoblastic lineage, which ultimately leads to active bone formation; and (5) the mechanical stimulation protocols used to maintain the integrity of the bone repair and their role in successful cell engraftment. Finally, a couple clinical scenarios are presented (non-unions and avascular necrosis—AVN), to illustrate how novel cell-based therapy approaches can be used. A thorough understanding of tissue engineering and cell-based therapies may allow for better incorporation of these potential therapeutic approaches in bone defects allowing for proper bone repair and regeneration. Fracture healing typically occurs uninterrupted during the first 6–8 weeks following an injury, although this process can be delayed by structural parameters such as the presence of thick cortices, which require more time to heal, as well as unfavorable mechanical and biological environments generated from excessive fracture site movement and/or gaps to general factors including aging, alcohol, tobacco, and steroid abuse and medical conditions such as infection, type 1 diabetes, anemia, and deficient nutrition , the generation of cartilaginous/periosteal bony callus, revascularization, mineralization, and resorption of cartilaginous callus, and finally bone remodeling. Within this cascade the homing of resident or systemically mobilized MSCs to the fracture site is of outmost importance and mainly mediated by signaling cascades such as stromal cell-derived factor (SDF)1/CXC chemokine receptor (CXCR) 4 signaling axis  can be used as the cellular component of engineered bone implants. However, technical difficulties associated with their harvesting, expansion into meaningful numbers and phenotypic maintenance undermine the benefits of using primary cells. Consequently, various types of stem cells have been largely proposed as a viable and easy source of osteoblast progenitors during the creation of engineered bone implants.Mesenchymal stem cells (MSCs) are multipotent adult stem cells that exhibit great differentiation potential into many different types of tissue lineages, including bone (osteoblasts), cartilage (chondrocytes), muscle (myocytes), and fat (adipocytes). Adult MSCs act as an inducible reserve force for tissue regeneration after injury  are currently the most commonly utilized and researched source of adult mesenchymal stem cells due to their relatively easy harvesting, high proliferative capacity, and established regenerative potential  or “viable allografts.” Bone marrow concentrates are originating from density gradient centrifugation and their MSC content is usually evaluated based on in vivo. In a case study, Mesimaki et al. performed maxillary reconstruction using a microvascular flap construct generated by combining autologous ADSCs, β-tricalcium phosphate and bone morphogenetic protein-2. At 36 months follow-up the implants were osteo-integrated without any adverse effects and the defect was fully reconstructed  derived from adipose tissue is composed of heterogenous cell populations including adipose-derived stem cells (ADSCs), vascular endothelial cells, vascular smooth muscle cells, preadipocytes, and haematopoietic origin cells . DPSCs were first isolated and characterized by Gronthos et al. within the dental pulp core and found to have similarities to BMSCs , umbilical cord tissue (UC) and placenta have been considered as alternative sources of MSCs. Moreover, UC and UCB are readily available sources of allogeneic MSCs for therapeutic applications due to the worldwide existence of both public and private cord blood (CB) stem cell banks. In various preclinical models, CB-derived MSCs have successfully enhanced bone regeneration in conditions of non-systemic and systemic bone loss  maintain the developmental potential for all three embryonic germ layers even after months of in vitro study was able to induce attachment, proliferation, and bone mineral synthesis of hESC-derived MSCs on a novel calcium phosphate cement-chitosan-RGD biomaterial /hydroxyapatite composite biomaterial , which are derived and reprogrammed directly from adult somatic cells , have the ability to give rise to every type of cell in the body and to propagate indefinitely. They were first developed by Takahashi and Yamanaka in 2006, through the lentiviral-based introduction of a defined set of reprogramming transcription factors , which induced a pluripotent state comparable to ESC. Consequently, iPSCs hold enormous potential for the entire field of regenerative medicine, as they possess a comparable pluripotency and differentiation potential as hESCs  have been established to support with best practices to generate and supply hiPSCs lines for basic and potential clinical research. Consequently, manufacturers have initiated projects aiming at generating iPSC products based on strict cGMP-compliant conditions to overcome some of these challenges, involving best practices for cell culture, documentation and quality control  and tricalcium phosphate being the most common.In one study, dense calcium sulfate (D-CaS), ultraporous tricalcium phosphate (beta-TCP) and porous silicated calcium phosphate (Si-CaP) biomaterials were used in rabbits. They observed rapid resorption of D-CaS, which left the defect site empty prior to new bone formation. High rates of dissolution have been found to cause biological reactions by inducing inflammation  which allow for the potential of bone formation similar to that of autografts, as well as excellent stiffness , the formation of surface coatings (such as polymers and glasses) , collagen, polyglycolide (PGA), and poly(ε-caprolactone) (PCL), are best known for their capacity to support tissue growth and remodeling prior to being resorbed. These biomaterials are hydrolytically stable as they contain ester bonds in their backbone, specifically short aliphatic chains, which allows them to be used as degradable biomedical constructs  (PLA) has been proven to be an effective polymer as studies have shown it can induce mature osteogenic phenotypes after 4 weeks of cell seeding. PLA-based biomaterials have shown adhesion and growth of human osteoprogenitors cells ultimately leading to bone healing  (PEGS) polymers also demonstrate optimization of their mechanical characteristics and bioactivity in BTE applications /tri-calcium phosphate (PCL/TCP) biomaterials by coating them with carbonated hydroxyapatite (CHA)-gelatin composite via biomimetic co-precipitation. They found an increased proliferation rate of cultured porcine BMSCs of about 2.3 times that of non-coated CHA-coated composites. In addition, the CHA-gelatin composite coated PCL/TCP biomaterials stimulated BMSCs osteogenic differentiation the most , vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1), and insulin-like growth factor 1 (IGF-1) being the major regulators of bone remodeling cascade. The therapeutic use of recombinant growth factors is based on the hypothesis that through appropriate signaling they induce and/or accelerate the bone healing process. However, only a number of recombinant growth factors (such as BMP-2 and BMP-7) have achieved commercial success due to limitations related to their safety, cost-effectiveness, low stability, short half-life and rapid deactivation of their actions in de novo bone formation , has been identified as an osteogenic factor in the repair of critical sized long bone defects and craniofacial bones , as well as significantly inhibited healing of tibial cortical bone defects (compromised intramembranous ossification), demonstrating how VEGF is essential to bone healing through chondrocyte intermediate and direct repair mechanisms  is a signaling molecule that plays a critical role in angiogenesis and the migration and proliferation of MSCs. Though there are multiple isoforms, PDGF-BB is considered the universal growth factor in this family due to its ability to bind to all isoforms of the PDGF receptor  until cleaved and activated by osteoclasts. Once active, TGF-β1 functions as a chemotactic agent to recruit local MSCs for osteogenic differentiation through the SMAD intracellular signaling transduction pathway. A study by Tang et al. used mice to demonstrate that TGF- β1 is necessary and responsible for inducing migration of BMSCs to skeletal remodeling sites in response to osteoclastic bone resorption. This study also showed that an induced mutation in the Igf1r−/−) showed a reduction in mature osteoblasts at the site of bone remodeling as well as a reduction in bone formation, demonstrating how IGF-1 signaling is necessary for bone remodeling. They were able to determine that when IGF-1 is released from the bone matrix during osteoclast activity, it is able to activate mTOR signaling to stimulate osteoblastic differentiation of BMSCs recruited by TGF- β1. Additionally, IGF-1 levels in the bone matrix were found to be low in rats with age-related reduction of bone mass  and age. In addition, bone segmental defects where bone loss cannot be compensated even by autologous bone grafting there is need to combine all different aspects of BTE including stem cells, growth factors, scaffolds and mechanical stimulation for effective bone regeneration  the type of cells and vehicle used to transport them, 2) final cell concentration, (3) immature or progenitor cell status, (4) time of therapy, (5) mode of administration and (6) availability of osteoblastic progenitors at the fracture site. We have discussed some of these factors above in relation with cells, biomaterials or growth factors employed. However, the availability of osteoblastic cells at the fracture site constitutes another critical factor, which in fact can be manipulated. Overall, recruitment  of endogenous MSCs to bone defect sites presents benefits and limitations when compared to exogenously administered autologous or allogeneic MSCs. Different strategies to induce homing of endogenous MSCs to bone defects are reviewed in Herrmann et al. . On the  the type final cein vitro before implantation. In this typical tissue engineering approach, the engineered implant demonstrated good integration with host tissue and functional clinical outcomes as early as 6 weeks post-surgery  in atrophic nonunion patients to healthy subjects in an effort to better understand the molecular and cellular mechanisms which lead to nonunions. Patients with nonunions were shown to exhibit a decreased pool of BMSCs and changes in the serum levels of chemokines and growth factors required for their recruitment and proliferation  has been explored as a viable source of BMSCs for the treatment of bone nonunions. BMAC is a heterogeneous cell mixture containing various cell types including hematopoietic and mesenchymal progenitors  along with EPC, monocytes, granulocytes and other cell types. Several studies have been reported using BMAC for nonunions, bone defects, distraction osteogenesis and others for potential complications of the therapy. The overall consensus of those studies is the clinical efficacy of BMAC inducing bone healing, its superiority to bone grafts, and reduced complications  is a disease which affects the younger population resulting in several complications. AVN can be idiopathic or secondary to chemotherapy, steroid therapy, trauma, chronic alcohol use, or sickle cell disease. Old reports have shown that more than 80% of patients with AVN develop end-stage osteoarthritis necessitating joint replacement  provides relief by decreasing the intra-medullary pressure, which alleviates the symptoms but does not impact progression of the disease  putative immunological rejection of allogeneic stem cell infusion in vivo and donor-related differences and (d) other translational difficulties. Specifically, for the treatment of delayed fracture healing and avascular necrosis bone defects, local administration of stem cell therapies in combination with specialized osteo-conductive biomaterials, osteo-inductive growth factors and adequate mechanical stimulation may further resolve more effectively and more rapidly these complicated orthopedic conditions. Collectively, a better understanding of the functional roles of stem cells in health, aging and disease will provide the foundations for the design of novel and advanced therapeutic strategies for skeletal disorders.A major challenge for the upcoming decade in the bone regeneration field is to elucidate the underlying cellular and molecular mechanisms of stem cell actions in bone defect therapeutics. In previous years, both pre-clinical and some clinical studies have shown beneficial effects, mainly with MSCs and osteoblastic progenitors in bone healing but the exact mechanisms of actions remain unclear. Although encouraging clinical results have been obtained by transplanting either uncultured MSCs or expanded MSCs, the exact dosage and route of application remain to be optimized and the fate of transplanted cells and their mechanisms of action need to be better monitored in larger clinical trials. On this basis, clinical applications of stem cell-based bone therapies are still limited due to various hurdles such as (a) ethical concerns related to ESCs and iPSCs therapeutic application, (b) proper phenotypic and molecular qualitative evaluation of stem cell products (c) laborious and expensive All authors contributed substantially to the conception or design of the work, the drafting of the work or revising it, reviewing for final approval, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.