Diabetic retinopathy (DR) is the most frequent microvascular complication of long-term diabetes and the most common cause of blindness, increasing morbidity in the working-age population. The most effective therapies for these complications include laser photocoagulation and anti-vascular endothelial growth factor (VEGF) intravitreal injections. However, laser and anti-VEGF drugs are untenable as a final solution as they fail to address the underlying neurovascular degeneration and ischemia. Regenerative medicine may be a more promising approach, aimed at the repair of blood vessels and reversal of retinal ischemia. Stem cell therapy has introduced a novel way to reverse the underlying ischemia present in microvascular complications in diseases such as diabetes. The present review discusses current treatments, their side effects, and novel cell-based and tissue engineering approaches as a potential alternative therapeutic approach. Diabetes mellitus is a chronic metabolic disease characterised by sustained hyperglycemia that leads to macro and microvascular complications . DiabeteDiabetic retinopathy (DR) manifests as a broad spectrum, particularly at the level of the retinal vasculature, and is responsible for 4.8% of the 37 million cases of blindness in the world according to the World Health Organization (WHO). The main risk factors for DR are high blood pressure, hyperglycemia, and the duration of diabetes. Studies have found consensus that there is a pathogenic link between hyperglycemia and the onset and progression of DR, while tight control of blood glucose can delay DR onset and progression. Some of the DR risk factors are gender, age at onset of the disease, ethnicity, cataract extraction, and hyperlipidemia . The durIschemia is characterised by the restriction of blood supply to tissue and organs, causing a shortage of oxygen and glucose which is needed for cellular metabolism and removal of metabolites . IschemiVaso-degenerative retinopathies, such as DR, can result in variable degrees of retinal vascular insufficiency and a profound loss of vision. Beyond the significant risk of depriving delicate neural networks of oxygen and nutrients, hypoxia also increases growth factor and cytokine expression. This can result in vascular leakage in the surviving vasculature and/or pre-retinal and papillary neovascularization. If these complications are left untreated, the responses to vascular stasis, ischemia or hypoxia can result in fibro-vascular scar formation or retinal edema and blindness ,7.Many diabetic patients may not experience any noticeable symptoms in the early stage of the disease. However, early detection of DR can help to prevent severe loss of vision and blindness. Different clinical signs of retinopathy include dot and blot retinal hemorrhage, the formation of microaneurysms, cotton wool spots, hard exudates, venous abnormalities, and growth of new blood vessels. There are also anatomical changes during DR that have been well-documented and include the formation of acellular capillaries, early thickening of the basement membrane, formation of microaneurysms, loss of pericytes and endothelial cells, and retinal neovascularization . DR diagDR can be classified by the clinical presentation either as non-proliferative DR (NPDR) or as proliferative DR (PDR). The first change observed in DR patients is a reduction in the retinal blood flow, which is followed by a loss of pericytes resulting in the development of micro-aneurysms, which may be associated with the appearance of retinal hemorrhages and hard exudates . These cAt any stage of the disease, DR can be associated with diabetic macular edema (DME), DME is defined as retinal thickening caused by vascular leakage and a build-up of fluid and proteins within two-disc diameters of the macular region. DME is the major cause of severe visual impairment in diabetic patients. Diabetic macular ischemia (DMI) occurs when small blood vessels close completely over time, resulting in poor blood flow. DMI causes the death of nerve cells in the macula responsible for fine vision, which is an irreversible process resulting in a permanent, untreatable central blind spot which decreases central vision .NPDR and DME are considered the most sight-threatening ocular complication. Many studies have demonstrated that prevention and modification of associated systemic risk factors are the critical steps for the treatment of diabetic retinopathy. The control of blood pressure, blood glucose and glycosylated hemoglobin levels, and lipid levels have all been associated with the reduction of the long-term risk of developing sight-threatening ocular complications. Much research has been carried out worldwide and has led to various novel therapeutic targets. The Early Treatment Diabetic Retinopathy Study (ETDRS) established pan-retinal and macular laser as the gold standard treatment for these complications . Laser photocoagulation and vitreoretinal surgery (vitrectomy) are the current surgical therapies that are effective in reducing the loss of vision and are useful for the late-stage disease of retinopathy but carry significant sight-threatening side effects . AlthougIt is important to mention that there are also other vascular mediators that has been shown to contribute to ocular angiogenesis including members of platelet-derived growth factor family (PDGF), fibroblast growth factor family (FGF), transforming growth factor-β superfamily (TGF-β1), epidermal growth factor family (EGF), hypoxia-inducible factors insulin-like growth factors, cytokines, matrix metalloproteinases and their inhibitors, and glycosylation proteins. Recent studies investigated new drugs such as Fovista (PDGF), X-82 (VEGF & PDGF), Squalamine lactate , and RG7716 (ANG-2 and VEGF) to target retinal neovascularization for future anti-angiogenic therapies. Full description of ongoing clinical trials of new drugs against retinal and choroidal angiogenesis are shown in Despite promising results with anti-VEGF therapy, some important issues should be considered. First, the requirement of multiple intravitreal injections can cause side effects, including cataracts, uveitis and retinal detachment . FurtherVascularisation is one of the most critical challenges to be addressed in tissue engineering so it can be clinically implemented. There are several important criteria that should be considered to achieve sufficient tissue-engineered vasculature. Cells should be located an optimum distance between a patterned vascular network and the surrounding tissue for proper oxygen and nutrient exchange. Vascular lumen must line up with the endothelium for adequate homeostasis and material exchange to enable quick integration with surrounding host tissue and development of a long-lasting vascular network .Within the vasculature hierarchy, the size of blood vessels can be significantly different which varies the design requirements and approaches for bioengineering . Small vAfter fertilization, the zygote divides mitotically and generates a morula (termed blastula in invertebrates). After the first series of cell divisions, the blastula/morula undergoes a spatial reorganization called gastrulation. This process starts with an infolding (primitive streak) of the single-layered blastula, eventually leading to three distinct germ layers known as the ectoderm (outer layer which produces cells of the epidermis and nervous systems), endoderm , and mesoderm  . The cirBlood vessels develop by a combination of vasculogenesis and angiogenesis. Vasculogenesis describes the de novo formation of blood vessels and is responsible for the formation of primary vessels during early embryonic development such as the dorsal aorta. It relies on the local differentiation of mesoderm-derived precursors of endothelial cells (PECs) into ECs that coalesce into primitive networks. Angiogenesis is the expansion of a pre-existing vessel network through a combination of sprouting, proliferation, and remodeling processes . In adulTo date, many cell-based approaches have used vascular endothelial cells as a potential source to regenerate blood vessels because of their ability to self-assemble into functional capillary network and integrate into host tissue. However, several concerns emerged. For instance, terminally differentiated ECs, such as microvascular endothelial cells, are limited in expansion potentials and it has been shown that isolation and purification of these populations need further invasive procedures. Additionally, not all ECs have the same functional properties due to the donor variability and tissue site septicity. Several in vitro and in vivo studies exhibited better functional phenotypes, higher vascular density, and more stable blood vessels when ECs co-cultured with other cell types such as pericytes, fibroblasts or MSCs compared to ECs alone ,41,42. TProgenitors of endothelial cells are known to be a promising stem cell source for vascular regeneration ,45. TheyIn vitro studies have suggested that there are at least two different types of EPCs: (1) early and (2) late outgrowth EPCs. Although both of these populations showed the capacity to promote vessel regeneration in different animal models, they illustrated different capabilities to differentiate into ECs and to physically contribute to new blood vessel formation. “Early EPCs”, also called non-colony forming EPCs, have myeloid/hematopoietic characteristics and share lineage traits with immune cells, specifically macrophages and monocytes. They are isolated from adult peripheral blood mononuclear cells (PB-MNCs) or human cord blood mononuclear cells (CB-MNCs), which are plated on Fibronectin-coated dishes for 48hrs to deplete the adherent macrophages and mature ECs. Then, non-adherent cells are removed and re-plated on Fibronectin-coated plates and VEGF containing medium. After 4–7 days, so-called “early EPCs” can be obtained . These cWith regards to in vitro studies, EPCs have shown to generate lumenized capillary-like structures on Matrigel and a partially functional capillary network inside a modified poly (ethylene glycol) (PEG) hydrogel in the presence of mural cells . The impStem cells may be sourced from blastocysts before implantation from day 5–7 of the embryo . Other types of stem cells can be derived from blood or other tissues postnatally (adult stem cells), as seen in . 72] Ea Ea72] EaResearchers have successfully derived ECs and SMCs from human embryonic stem cells (hESC) lines. Several different approaches have been used to differentiate SMCs from hESCs. Endothelial progenitor cells such as CD34+ cells have been isolated either from spontaneous differentiation of embryoid bodies (EBs) or from co-cultures and then induction with factors such as TGF-β1, PDGF-BB, and retinoic acid ,76. OtheiPSC cells are a type of PSCs that can be prepared by re-programming adult somatic cells into stem cells, initially illustrated by Yamanaka in 2006 . iPSC teIt has been shown that iPSC-derived Ecs are able to incorporate into damaged vasculature of ischemic tissue and improve function ,87. HoweHuman adult MSCs, identified in different tissues such as bone marrow and adipose tissues, are a type of adult multipotent stem cell with a more restricted differentiation potential and limited self-renewal ability . StudiesThere are several 2D and 3D assays to develop tube-like structures. In 2D wound-healing assay, cells are cultured in a monolayer and, when cells are confluent, a “wound” is created and images captured from the beginning and at specific intervals during cell migration until the wound is closed. This method was one of the very early methods to study the directional cell migration in vitro . More reSeveral studies have shown that angiogenic stimulation can occur in vivo by using a variety of cytokines and gene-based and cell-based approaches. Pre-clinical studies, for instance, have shown successful angiogenic response in a rabbit hind limb ischemia model to form collateral vessel formation by intra-arterial injection of VEGF ,108,109.Endothelial cells can incorporate themselves into the implanted scaffold and form vascular self-assembly structures in vivo. As previously discussed, using co-culture system can decrease apoptosis and increase implanted cell survival . In vivoThe decellularization method is one of the most promising techniques for tissue regeneration in which the extracellular matrix (ECM) is isolated from native cells to produce a natural scaffold. Decellularized ECM maintains its mechanical, structural, and biochemical cues which can then be recellularized to produce a functional tissue mimicking the natural organization of specific tissue. Among different decellularization methods, the method that does not cause disruption of vascular integrity is the most suitable for cellular repopulation. Furthermore, proper endothelial repopulation of acellular scaffold is crucial to prevent microvascular coagulation stimulated by the collagenous matrix. This approach is one of the promising methods currently to generate robust perfusable microvasculature, though new microfabrication approaches are quickly developing ,115.Recently, the use of microfabrication techniques to produce controlled and reproducible patterns of vasculature have been widely investigated. Although these techniques are not ready for clinical applications, they are crucial developments to shape future directions. Durable engineered vasculature has important features including high mechanical properties to prevent rupture and accommodate flow with highly pulsatile pressure, low thrombogenicity, and high regenerative integration with the host tissue ,116.Recent advances in microfluidics and 3D bioprinting have been used to generate vascular constructs in tissue engineering applications. Microfluidic applications are divided into three different categories including sacrificial molding, micropatterning using soft lithography, and in vitro self-assembled endothelial cells . All of 3) are great platforms for disease modeling and drug testing. However, it is unknown if these constructs can further enhance stabilization and function of microvessels after in vivo transplantation. A final approach is to utilise endothelial cell ability to self-assemble into microvascular networks by applying interstitial flow to extracellular matrix-containing combinations of endothelial cells and mural cells within microfluidic chambers. These small-scale constructs (<1 mmLaser-based degradation of hydrogels, biomaterials, and other soft interfaces have recently garnered attention as a technique for obtaining high-resolution control over user-defined architectures within 3D tissue constructs and acellular platforms. Another method to generate a vascular network is to selectively degrade regions within a bulk crosslinked ECM. In this technique, a biomaterial is first crosslinked into a hydrogel, and then a laser is used to degrade the synthetic material locally. This technique was first used to fabricate channels with a diameter as small as 3 μm, though it was difficult to flow HUVECs through these channels without clogging . DespiteThe proper formation of blood vessels is critical for the generation and continued function of tissues and organs during embryonic development and throughout life. Vascularization is the key challenge that hinders the clinical application of tissue-engineered products and, thus, the greatest obstacle to successful transplantation. To date, various strategies have been developed to generate vascular constructs at clinically relevant scales. However, the generation of vascular networks that can mimic the complexity, ultrastructure, geometry, and biochemical cues remains a challenge. Moreover, timely-mannered integration of engineered constructs after implantation is critical for optimum functionality of the implanted construct. To promote this, novel approaches have cultivated prevascularized constructs that can rapidly inoculate the implanted vascular construct to the host tissue, enhancing the survival of implanted constructs. Further, advanced imaging techniques enable the viewing and monitoring of the detailed structures in the integrated constructs over time. This derives from the current capacity of some optical imaging methods in clinical or preclinical research for micro-structural imaging or functional imaging ,132,133 However, some of the drawbacks of these novel approaches are also important to be taken into consideration including tumourigenity, cell migration, graft rejection, immunogenity, and complexity of the host immune response . The imm


The increased worldwide prevalence of obesity, insulin resistance, and their related metabolic complications have prompted the scientific world to search for new possibilities to combat obesity. Brown adipose tissue (BAT), due to its unique protein uncoupling protein 1 (UPC1) in the inner membrane of the mitochondria, has been acknowledged as a promising approach to increase energy expenditure. Activated brown adipocytes dissipate energy, resulting in heat production. In other words, BAT burns fat and increases the metabolic rate, promoting a negative energy balance. Moreover, BAT alleviates metabolic complications like dyslipidemia, impaired insulin secretion, and insulin resistance in type 2 diabetes. The aim of this review is to explore the role of BAT in total energy expenditure, as well as lipid and glucose homeostasis, and to discuss new possible activators of brown adipose tissue in humans to treat obesity and metabolic disorders. Brown adipose tissue (BAT) was considered, for several years, to be present only in newborns and small mammals to generate heat through non-shivering thermogenesis as protection against hypothermia. However, the abundant amount of active BAT in children declines rapidly after puberty. The exact amount (volume) of active BAT in adult humans remains highly variable, but the prevalence of brown adipose tissue in adults was estimated at 6.97% based on recently published results from a systematic review and meta-analysis . The firThe increased worldwide prevalence of obesity has prompted the scientific world to search for new possibilities to deal with weight gain . ObesityBAT is a type of tissue designed for maintaining body temperature higher than ambient temperatures through heat production, primarily via non-shivering thermogenesis. This process is mediated by the expression of uncoupling protein 1 (UCP1) within the inner membrane of the abundant mitochondria . DespiteDue to its unique UPC1, brown adipose tissue has been acknowledged as a promising approach to increase energy expenditure . In otheThe protective role of BAT, in terms of its metabolic consequences, prompted the molecular exploration of brown adipocyte differentiation. The most relevant molecular factors involved in brown and white adipose tissue formation are peroxisome proliferator-activated receptors (PPARs) . PPARγ hThe aim of this review is to explore the role of BAT in whole-body energy expenditure and lipid and glucose homeostasis and to discuss new possible activators of brown adipose tissue in humans to treat obesity and metabolic disorders.Brown adipose tissue (BAT) and white adipose tissue (WAT) contribute to total adipose tissue in humans. Despite having similar structural components (adipocytes), the functions of both fat tissues are different. White adipose tissue stores energy, while brown adipose tissue generates body heat. Brown adipose tissue consists of brown adipocytes  and lipids that are located in multiple small droplets, giving brown adipocytes a multilocular histology  with a cThe traditional understanding of white fat tissue as passive energy storage is no longer justifiable. In recent years, adipose tissue has been recognized as a complex, highly active metabolic and endocrine organ , with spThermogenesis is generally described as any metabolic process that releases heat, so whole-body thermogenesis is a counterpart of total energy expenditure (EE) . Body te11C-acetate, which evaluated the Krebs’ cycle rate, more accurately verified the role of BAT in cold-induced thermogenesis in humans [15O2 estimated BAT energy expenditure at a level of 15–25 kcal/day [The gold standard method for identifying and assessing BAT volume is 18FDG (18 fluoro-deoxy-glucose) PET/CT , but BATn humans , showingn humans ,47 withon humans . Studieskcal/day . A seconkcal/day . These akcal/day . The samkcal/day ; such ankcal/day  support kcal/day . The appkcal/day .Recent discoveries have shown the different mechanisms for facultative energy expenditure in BAT, including the futile cycles based on creatine  and succNegative energy balance is not the only factor in favor of using brown adipose tissue for the treatment of obesity; an additional significant feature is the ability of BAT to alleviate insulin resistance and disturbance in glucose homeostasis. Results from experimental studies showed that active BAT is inversely correlated with the body mass index (BMI), which additionally confirms the relationship between BAT and body mass . PatientA less frequent prevalence of BAT was noted in obese subjects compared to lean individuals, indicating the thermogenic effect of brown adipose tissue on body weight reduction . This isObesity is characterized by the chronic low-grade activation of the innate immune system. In this respect, macrophage-elicited metabolic inflammation and adipocyte–macrophage interactions have a primary importance in obesity . AdipocySurprisingly, in brown fat depots, the number of macrophages is rather low  or even Brown adipocytes exhibit the intrinsic ability to impair the inflammatory profile of macrophages, while white adipocytes enhance this profile. This suggests that brown adipocytes may be less prone to adipose tissue inflammation, which is associated with obesity .Recently, Fisher et al., demonstrated that the expression levels of almost all macrophage marker genes (M1 and M2) were much lower in the brown fat of mice acclimated to room temperature (middle-aged and young mice) than in the brown fat of thermoneutral mice (30 °C) . MoreoveThe majority of macrophages found in the brown fat of thermoneutral mice are organized into multinucleate giant crown-like structures. The authors hypothesize that the macrophages found in thermoneutral brown fat perform their conventional (but likely not their only) function: phagocytosis and degradation of dead cells. Brown fat macrophages thus orchestrate tissue remodeling and enable the maintenance of metabolic homeostasis in tissue analogically to the situation in WAT .The brown fat of thermoneutral mice retains full competence during the process of cold acclimation. Thus, profound macrophage accumulation does not influence the thermogenic recruitment competence of brown fat.The activation of brown adipose tissue is triggered by cold exposure. Lower temperatures are detected by skin receptors, which convey signals via the wider neuronal network, including the hypothalamus as a key regulator of body core temperature, through the spinal cord, and finally to the peripheral sympathetic nervous system (SNS) of BAT. Noradrenalin is released after the activation of SNS; it then binds to adrenoreceptors (mainly the B3 receptor) and the lipolysis process of brown adipocytes is initiated. Fatty acids from triglyceride (TG) lipid droplets are the main source of oxidation by UCP1 in brown mitochondria. The reduced amount of TG needs to be restored mainly through the uptake of glucose, albumin-bound free FA, and TG-derived fatty acids from LDL and chylomicrons in the plasma. Findings from animal studies confirm the involvement of BAT in total energy expenditure and TG clearance and metabolism . MoreoveThe use of the 18F-FDG tracer in imaging studies with PET confirmed glucose uptake by human BAT. The 18F-FDG tracer is a glucose analogue that is transported into cells by the same transporters as glucose. The presence of Glucose transporter type 4 GLUT-4 and GLUT-1 was first identified in brown murine adipocytes, suggesting both the insulin-dependent and insulin-independent uptake of glucose by the tissue  In activThe insulin-mediated FDG uptake by BAT suggests that the GLUT-4 transporter participates in glucose uptake in human BAT. Consequently, BAT could be considered as an insulin-sensitive tissue ,82. StudThe decreased activity of BAT, through impaired glucose uptake in resting conditions and reduced glucose clearance from serum, may predispose an individual to T2D. Recently published epidemiological outcomes indicated an association between increased glycosylated hemoglobin and an increased prevalence of diabetes with higher outdoor temperature . MoreoveAnother study has shown decreased BAT glucose uptake rates in overweight and T2D subjects vs. young healthy subjects with no reduced uptake of non-esterified fatty acids and thermogenic activity under cold stimulation .Outcomes from a previous publication estimated a mean total volume of 18FDG-positive BAT at 150 mL in healthy adults , which cThe most common method of activation for brow adipocytes is cold exposure . The outCold exposure certainly plays the most significant role in increasing the metabolic rate via BAT activation. However, studies in both animals  and human-3 long-chain polyunsaturated fatty acids enhanced thermogenesis via the activation of brown adipose tissue [Data from experimental studies indicate that some dietary supplements can increase the metabolic rate via the thermic effects caused by BAT; once such supplement is capsaicin derived from chili peppers. This supplement enhanced thermogenesis, accompanied by a reduction in body fat in both animals  and humae tissue ,118. More tissue ,120. Thee tissue .Classical BAT depots are typically found in animals in interscapular, cervical, peri-aortic, peri-renal, intercostal, and mediastinal areas . The broIn adults, brown-like adipocytes with typical brown adipocytes are scattered among white adipocytes in supraclavicular regions  and are +) progenitor cells, similar to skeletal myocytes [−) progenitor cells, much like white adipocytes [Classical brown adipocytes originate from myogenic factor 5 positive  . These tSkeletal muscles during physical activity are the source of another protein named irisin, which has a significant browning effect on white adipocytes in mice. Irisin is myokine-cleaved from fibronectin type III domain containing protein 5 (FNDC5), which is released by the exercising muscle. In animal models, an increase in irisin during exercise or exogenous administration resulted not only in an increase in WAT browning, but also in a significant reduction in body weight and an improvement in glucose tolerance . OutcomeThe function of activated brown adipose tissue does not only promote negative energy balance, but also alleviates metabolic complications such as insulin resistance, dyslipidemia, and disturbances in glucose homeostasis. There is still debate around how to maintain and activate the significant number of brown adipocytes needed to exert anti-type 2 diabetic effects. Therefore, future studies will involve the search for new BAT activators.Currently, the best-explored BAT activators are cold exposure and sympathetic nervous system agonists. In terms of the metabolic benefits resulting from BAT activation, the most important future perspective is to investigate the new molecular and environmental factors elucidating BAT activation, as well as the browning of WAT. The re-activation and recruitment of BAT in obese individuals is especially important because such individuals will receive the most benefit. It remains challenging to identify BAT activators that will be effective under conditions other than cold exposure. Moreover, the role of diet and nutrients is worth exploring to find new methods of BAT activation. Thus far, it has been shown that the stimulation of transient receptor potential channels via some food ingredients, such as capsinoids and the catechins found in green tea, exert anti-obesity effects by the activation and acquisition of new brown adipocytes. Another major goal for future research is the identification of brown adipokines or batokines that could be candidates for drug development to treat obesity or metabolic disease.The influence of BAT on energy expenditure is underestimated due to the small volume of BAT as measured using 18FDG PET. The accurate measurement of BAT volume is a major limitation, especially in individuals with obesity and T2D. The development of novel imaging methods for the precise quantification of BAT volume and activity is required to assess the true potential of targeting BAT thermogenesis to prevent or treat metabolic disorders.


Firearm dealer density is correlated with firearm interpersonal violence, but no quasi-experimental studies have assessed whether changes in dealer density lead to changes in firearm self-harm injuries and deaths. We assessed whether openings of firearm dealers are associated with short-term changes in local firearm self-harm injury rates.We identified 718 openings of firearm dealers in California using licensing data, 2014–2016. We defined exposure regions based on aggregations of zip codes defined by proximity to firearm dealer openings and matched each opening to four control regions on time and determinants of firearm injury. We applied a differences-in-differences approach to compare rates of firearm self-harm, in the month before and after each opening, in places with and without openings.Firearm dealer openings were not associated with acute, local changes in firearm self-harm relative to places without openings (ratio of rate ratio: 0.90 [95% CI:0.68–1.19]). Results were robust to numerous sensitivity and secondary analyses.We found no associations of firearm dealer openings with acute, localized firearm self-harm deaths and injuries. Our focus on acute, local effects; broad availability of dealers and firearms; durability of firearms; or strong confounding-control may explain these null findings. Firearms are a major means of self-harm and the most common method of suicide in the United States, accounting for approximately 25,000 suicides and 4,000 intentional self-harm injuries in in 2018 , 4. WhilLocal retail environments are modifiable features of communities that shape health-related behaviors , 7. For To our knowledge, only one previous study has examined firearm dealers and self-harm. Chao and colleagues found a positive association of state-level firearm dealer density with firearm suicides , while PWe exploit variation in the timing and location of firearm dealer openings with a quasi-experimental, differences-in-differences design to investigate whether acute increases in firearm dealer density are associated with short-term increases in firearm suicides and self-harm injuries. This approach strengthens confounding control while focusing on effects immediately following and in close geographic proximity to openings.We used a quasi-experimental, difference-in-differences design , 25. FirIn California, all firearm dealers are required to hold federal firearms licenses (FFLs). We compiled monthly data on California FFLs from the Bureau of Alcohol, Tobacco, Firearms, and Explosive’s (ATF) Federal Firearms Licensee Listings. Records are comprehensive, publicly available as of 2014, and include the geographic address of the licensee, license type, and month and year of license issuance. Multiple types of licenses exist; we restricted our study to FFLs that transfer firearms and ammunition, including licensees who transfer firearms but lack storefronts. We excluded licenses corresponding exclusively to “manufacturing”, “importing”, or “destructive devices other than firearms.” We assumed that an FFL “opened” in a month where a new license appeared for a physical address at which no licenses existed in the month prior. FFL data for September and October 2015 were not recorded due to a government shutdown; we treated these data as missing completely at random .th Revision Clinical Modification, 10th Revision Clinical Modification, or 10th Revision external cause of injury code and the residential zip code of the patient/decedent , 9We identified control communities by matching the zip code of each opening to zip codes without a firearm dealer opening on key determinants of firearm self-harm at baseline : month aWe constructed the analytic database at the level of the exposure region (aggregations of zip codes) and month (before or after the opening). Because FFLs are common, we hypothesized that openings would only influence small geographic areas in their immediate vicinity. Lacking evidence on the durations or distances over which FFLs might have effects, we defined a unique “exposure region” for each FFL opening (or control community) by combining the zip code of the FFL (or matched control zip code) with other zip codes with centroids within 5 kilometers (KM) of the FFL  period of one opening (event B) overlapped with the “after” (exposed) period of an earlier opening (event A). In this situation, we excluded the overlapping zip codes from analyses of event B . We also excluded rates that were extreme outliers (greater than 20 standard deviations from the mean).We controlled for place-specific time-varying covariates that might be related to acute variations in firearm self-harm. These were monthly county-level unemployment rate (Bureau of Labor Statistics), precipitation, and average temperature (WestMap PRISM Climate Mapping Program).We used negative binomial regression to estimate associations of FFL openings with firearm self-harm using the lme4 package in R. Statistical testing of the dispersion parameter indicated that a negative binomial model fit the data well. We modeled the count of firearm self-harm injuries as a function of time (month before versus after the opening); an indicator of exposure to the opening (versus unexposed); an interaction between time and exposure status, an indicator for the transition from ICD-9-CM to ICD-10-CM on October 1, 2015; the time-varying place-specific covariates; random effects intercepts on exposure regions nested within cities; and an offset for the population of the exposure region. The primary association of interest was the exponentiated coefficient on the interaction term: the ratio of rate ratios (RRR) measuring the before-after change in firearm self-harm for places exposed to openings versus unexposed places. See Ethical review was conducted by the California Health and Human Services Agency and the University of California, Berkeley Committees for the Protection of Human Subjects.We conducted sensitivity tests varying the sizes of the time units and geographic areas of analysis. In addition, although the month during the opening includes a mixture of both time before and after the opening itself, we conducted sensitivity tests comparing firearm self-harm rates in the month during versus the month prior to openings, to evaluate whether any associations could be detected more immediately than in the month after openings.In secondary analyses, we tested the hypothesis that the effects of FFL openings would be strongest for regions with no other nearby FFLs (in the same zip code) by restricting analyses to these regions. Additionally, because previous research identified variation in the associations of FFLs with firearm violence by urbanicity –23, we tWe measured our level of statistical power to detect effects, using an anticipated RRR of 1.15, assuming that FFL opening effects would be substantially smaller than those for gun shows (observed RRR 1.70 ). This aWe identified 718 FFL openings during the study period. After excluding outlier rates and applying overlap exclusions, we included 580 openings with 2,272 controls in the main regression analyses . The priResults of sensitivity analyses varying the sizes of the time units and geographic areas of analysis are presented in In secondary analyses, restricting to openings without other nearby FFLs, no meaningful associations were observed . OpeningTo our knowledge, this is the first study to assess whether openings of FFLs were associated with short-term increases in firearm self-harm in the communities where these openings occur. We found no meaningful overall associations of FFL openings with firearm self-harm. For rural areas and regions with no other nearby firearm dealers, results were suggestive of potential reductions in firearm self-harm associated with FFL openings, but estimates were too imprecise to draw conclusions.Given our focus on local, short-term effects, we anticipated modest effect sizes. However, broad availability of firearms and FFLs may mean that changes in exposure to a single FFL have negligible impacts, especially because firearms are durable goods that can be used without replacement for long durations. Although FFL data do not include sales volumes, we sought to identify the highest-impact places by testing effects for localities without any other nearby FFLs. Results of this analysis were also null, which strengthens our confidence in the results.Our hypothesis that FFL openings would lead to increases in firearm self-harm was based on prior studies that identified positive associations of firearm dealer density with firearm suicide, homicides, robberies, gun-related crimes, and firearm ownership –23. HoweGiven that all studies involve tradeoffs and untestable assumptions , this stOur focus on California is important in interpreting the findings. California has some of the most restrictive firearm regulations in the country. For example, all firearm transfers in California are legally required to occur through an FFL, including transfers between private parties, and involve a 10-day waiting period. Although adherence and enforcement are undoubtedly imperfect, regulatory differences like this may mean that illegal or harmful activity leading to FFL-associated injuries may be less likely to occur in California. Supporting this, one prior study of Californians found that gun shows were associated with increases in firearm injuries for shows that occurred in Nevada, but not shows in California . One proSeveral additional limitations of this study must be noted. First, residual confounding is possible, although we sought to minimize bias through our study design. Although we controlled for unemployment rates, openings may reflect changes in the economic environment not captured by unemployment rates. Second, our identification of FFL openings relies on changes in dealer license status; delays or inaccuracies in reporting of license status or in correspondence between license issuance and dealer opening may have resulted in misclassification. Third, although we tested exposures over multiple geographic distances and time spans, it is possible that we have missed the most relevant exposure characterization. We estimated the overall association of FFL openings with firearm self-harm but dealer size, type , or sales volume may modify any associations. Fourth, we did not examine firearm homicide or assault outcomes because, given evidence that time and distance from firearm purchase to crime are often long , we hypoIn summary, we found no associations of firearm dealer openings with local, acute changes in firearm self-harm in California. This study adds to a small literature on firearm dealers as a feature of the local retail environment. Our findings differ from the five previous studies that have identified associations of firearm dealer density with firearm crime or injuries, possibly because we examined acute effects on firearm self-harm, or because our study design affords greater confounding control. Further research is warranted to determine the reasons for these differing findings, as well as the most relevant measures of firearm dealer availability.S1 File(DOCX)Click here for additional data file.


Exosomes, nanometer-sized lipid-bilayer-enclosed extracellular vesicles (EVs), have attracted increasing attention due to their inherent ability to shuttle proteins, lipids and genes between cells and their natural affinity to target cells. Their intrinsic features such as stability, biocompatibility, low immunogenicity and ability to overcome biological barriers, have prompted interest in using exosomes as drug delivery vehicles, especially for gene therapy. Evidence indicates that exosomes play roles in both immune stimulation and tolerance, regulating immune signaling and inflammation. To date, exosome-based nanocarriers delivering small molecule drugs have been developed to treat many prevalent autoimmune diseases. This review highlights the key features of exosomes as drug delivery vehicles, such as therapeutic cargo, use of targeting peptide, loading method and administration route with a broad focus. In addition, we outline the current state of evidence in the field of exosome-based drug delivery systems in systemic lupus erythematosus (SLE), evaluating exosomes derived from various cell types and engineered exosomes. Autoimmune diseases are among the leading causes of morbidity and mortality associated with chronic disease worldwide, especially in women, who comprise more than 90% of affected patients ,2. AutoiExosomes are lipid-bilayer-enclosed extracellular vesicles released by many cell types in both normal and pathological conditions, and which transport nucleic acids, lipids and proteins between cells ,19. As dCurrent evidence shows that exosomes are able to modulate the immune system, as shuttles for antigen presentation ,21,22,23Due to their suitable proprieties, as well as their known therapeutic effects, exosomes are attracting the curiosity of researchers to develop exosome-based drug delivery systems. This review centers first on the biogenesis of natural exosomes and their function in SLE. Next, we will narrow our focus to identify the key components enabling successful exosome-based drug delivery, to unravel the potential of exosomes for disease treatment, finally covering new insights in exosome-based drug and gene delivery for future clinical use in SLE. Extracellular vesicles are small spherical lipid bilayer-coated vesicles, secreted by multiple cell types and present in many body fluids, which mediate intercellular communication ,34. NowaThe most broadly studied EVs are exosomes, 40–150 nm in diameter, endosome-derived and originating from intraluminal vesicles (ILVs) that reside inside multivesicular bodies (MVBs) . NucleicWhen released to the extracellular space, exosomes interact with their acceptor cells in different ways. Information transmission can occur at the cell surface without delivering any cargo (surface binding), as occurs during immune responses ,42, but Indeed, due to their involvement in normal and pathological cellular physiology, EVs have been recognized as good biomarkers in a wide variety of diseases including cancer, cardiovascular diseases, nephropathies and autoimmune diseases ,62,63,64Systemic lupus erythematosus is a prototypic autoimmune disease characterized by diverse immune disturbances. One of the serologic hallmarks of SLE is the production of antibodies to nuclear molecules (antinuclear antibodies (ANA)) ,4. TheseSeveral reports indicate higher levels of circulating IC-carrying MPs in SLE; as an example, Ullal et al. demonstrated that MPs display DNA and nucleosomal molecules in an antigenic form and could represent a source of ICs in SLE . Another+ in patients with SLE [Given their composition and immune properties, EVs act as proinflammatory mediators in SLE . In one with SLE . Anotherwith SLE . Finallywith SLE . These fSeveral studies reported EVs as reliable biomarkers of disease activity beyond their role in regulating immune responses, offering a valuable complement to classical laboratory markers ,78. NielMany recent studies highlight exosomal-derived miRNAs levels to identify distinct miRNA to discriminate LN. Our group revealed increased urinary exosomal miRNAs levels in patients with SLE, discriminating LN , and in In summary, quantity and phenotype of circulating EVs may be useful as novel biomarkers of activity and progression of SLE, providing a new therapeutic approach.Research interest in exosomes has grown dramatically during the last few years due to their unique properties. Transport inside exosomes allows concurrent intercellular communication by delivering different signals simultaneously. Unlikely free circulating soluble factors, they have the ability to release large amounts of functional molecules to recipient cells . MoreoveThe potential use of exosomes in therapy is based on the cargos they deliver to recipient cells. Endogenous cargos with known beneficial properties or artificially modified molecules can be loaded using a range of methods. Likewise, different exosome administration routes for therapy purposes have been described, with novel methods being developed during recent years .Since of exosomes have an inherent ability to carry different types of active molecules, they can be biologically or chemically loaded to deliver enhanced or broadened therapeutic compounds, representing an informed way to treat many diseases. Research as drug delivery vehicles is based mainly on the transfer of small RNAs, although other possibilities are being explored.Exosome based-miRNA therapy is the most developed area so far, since miRNAs function as gene expression regulators and have been implicated in the pathophysiology of numerous diseases. In addition, exosomal ability to carry therapeutic miRNAs has been extensively analyzed, demonstrating high effectiveness . This thSimilarly, mesenchymal stem cell (MSC)-based therapies have also demonstrated protective effects by promoting tissue repair, apoptosis and fibrosis inhibition or stimulation of cell proliferation ,92. MSCsPreliminary studies of the time laid the groundwork for pursuing exosomes as the best drug delivery system option for miRNA therapy, and paved the way to develop multiple strategies for refinement of methods and for direct targeting of exosomes to cell types.Small RNA interference (siRNA) has become a promising therapeutic strategy for knocking down targeted genes; however, their use as therapeutic compounds has limitations, as they are extremely hydrophilic and easily degraded . MoreoveThere is emerging interest in long non-coding RNAs not only as biomarkers but also as therapeutic agents due to their ability to interact with miRNAs. lncRNAs are naturally carried by EVs and are been increasingly documented as key mediators of EV biological effects . TherapeAs shown before, exosomes are promising targets for therapeutic delivery based on small nucleic acids such as siRNAs and miRNAs, which are successfully loaded inside EVs. However, the use of exosomes as drug delivery systems of exogenous DNAs remains poorly researched despite the widely reported presence of different DNA species inside exosomes ,117. LoaIn addition to different nucleic acids, exosome transported proteins are also being used as therapeutic cargos, albeit less commonly ,124. AspApart from the natural cargos of exosomes , their excellent biological properties have driven their use as drug delivery systems of synthetic compounds  have been attempted to improve the efficiency and specificity of EVs delivery. Exogenous loading after EV isolation, which can be divided into passive and active processes, includes incubation as a passive option and sonication, extrusion, freezing and thawing cycles, electroporation and chemical-based transfection as active processes where the use of electric field or surfactants are required for EV membrane permeabilization. Endogenous manipulation refers to introducing the cargo of interest into EV-producing cells, commonly transfecting cells with expression vectors. In addition, modification or attachment of molecules and receptors to EV surface are key ways to enhance delivery of targeted therapy .Passive loading of EVs can be simply performed by incubating exosomes with the cargo of interest. The differing concentration inside and outside the EVs will drive diffusion of hydrophobic cargos through EVs lipid-bilayer. Several chemotherapeutic drugs such as doxorubicin and paclitaxel have been loaded into exosomes by incubation ,134, demSaponification allowed up to 11-fold higher drug loading of large or hydrophilic compounds like porphyrins compared to passive methods . In thisAn additional loading technique is to apply sound energy with a sonicator to exosomes solution, which induces mechanical shear forces affecting exosome membrane integrity and enhancing the incorporation of the cargos present in the solution to vesicles . This prExtrusion is a process by which exosomes are loaded by membrane disruption when extruded through small polycarbonate porous membranes, allowing to incorporate cargos present in the solution to the vesicles . DespiteThis method consists of freezing and subsequently thawing a solution of vesicles with the cargo of interest. The mix is incubated at 37 °C, frozen rapidly at −80 °C and then thawed at room temperature, repeating at least three times. One inconvenience of this method, however, is aggregation of vesicles into large-sized particles ,138.Permeabilization of EV membrane by applying an electrical field is one of the most common techniques employed to enhance uptake of the cargo of choice. Disruption of the phospholipid bilayer of EVs allows the hydrophilic compounds to be diffused into exosomes such as small DNAs ,139, miRUse of chemical reagents for siRNA loading into exosomes has also been described. Wahlgren et al. employed liposome-based transfection reagent to introduce MAPK-1 siRNA into exosomes by incubating the mixture for 10 min at room temperature . LikewisAnother interesting approach is the generation of engineering cells to produce loaded exosomes with specific cargos. One approach is transfection of donor cells to overexpress a particular gene, allowing the generation of specific gene-loaded exosomes during their biogenesis. Jiang et al. investigate the therapeutic effect of TSG-6 modified MSC-derived exosomes on a mouse full-thickness wound model, providing evidence of a possible mechanism to prevent from scar formation . In addiIn addition to isolating, loading and/or modifying exosomes, selecting the exosome administration route and dose are also important parameters to consider to achieve clinical relevance in the future. In this respect, a key challenge is enhancing the biodistribution, stability and therapeutic effect of exosomes. In the following subsection we summarize some of the most frequently used exosome administration routes. Systemic injection is one of the most commonly administration routes used in exosome therapeutics ,106. AmeSeveral studies provide evidence that exosomal subcutaneous injections are effective in several pathologies ,158, butIntranasal injection provides a practical option for administration of therapeutic compounds to the brain. One of the first studies reported that curcumin-loaded EL-4-derived exosomes were efficiently delivered to microglia cells by intranasal administration, showing protection against lipopolysaccharide (LPS)-induced brain injury . In addiAlthough not commonly used as intravenous injection, intraperitoneal delivery of exosomes is another administration route option . InteresIn addition to the administration routes previously described, oral treatment offers a simple non-invasive route of exosome delivery. This method, like others summarized above, has been employed to administrate chemotherapeutic drugs such as curcumin, resulting in three- to five-fold higher levels of curcumin in several organs compared to free delivery of the compound . RegardiFrom our literature review, we found a paucity of data comparing exosome loading by these different methods. Thus, it will be a vibrant area of research to assess the optimum doses required in vivo. In addition, the efficacy and safety of cargo-exosome delivery should be characterized in association with other available methods for gene and drug delivery to discover significant breakthroughs as well as find innovative applications of this novel approach.The properties of exosomes alone are not sufficient to guarantee the specific delivery of drugs and the enrichment of drugs in disease tissues. Indeed, a wealth of literature supports that exosomes need targeting strategies to improve the therapeutic effect of drugs .Targeted delivery of exosomes with increased biodistribution and specificity can be assessed by transfection of parent cells. The fragment is transfected by fusing the ligand of interest to the coding sequence of exosomal signaling peptide, allowing the protein of interest to attach to the vesicle surface . The mosDirect functionalization of exosomes is another option for exosome surface modification. Functionalization strategies tested, include covalent and non-covalent chemical. Covalent modification includes copper-catalyzed azide-alkyne cycloaddition (CCAAC) click chemistry, the reaction of an alkyne and an azide chemical group to form triazole linkage . The proAlong with the surface modifications described, stable modification of the exosome surface by non-covalent alteration of the EV membrane has been also developed and is summarized below. Multivalent electrostatic interactions involve binding highly cationic species to negatively charged groups located in the exosome membrane . HoweverDevelopment of modified exosomes has an auspicious future in drug delivery research. Innovative targeting moieties should be designed to promote enhanced delivery to a specific cell type. Although researchers have made impressive progress in the modification of engineered exosomes, most of the above-mentioned techniques are based on the modification of exosome donor cells. These changes may affect the protein composition and function of exosomes. Therefore, it is necessary to explore new techniques for exosome-targeted modifications to minimize changes in their composition. Maintaining progress in these areas will bring breakthroughs in the field and transform exosomes from promising candidates into smart nanoscale therapeutics.Both in themselves and as vehicles of drug and gene delivery, exosomes are under active development as therapeutic agents ,186. ExoThe use of EVs as a cell-free therapeutic alternative offers several distinct advantages over parent stem cells. A major advantage is that EVs, depending on their source, may be less immunogenic than their parental cells, likely due to a lower abundance of transmembrane proteins such as MHC complexes . Unlike Immune-therapeutic exosomes include naturally occurring exosomes, exosomes secreted by modified cells, and exosomes loaded with exogenous cargos .Several studies have shown that EVs from various cell sources have a therapeutic effect through their intrinsic content , which iOne major role of MSCs is to suppress proliferation and function of cells in both innate and adaptive immunity responses . Thanks MSC-EVs can exert immunosuppressive effects on T cells , inhibitLoading therapeutic cargos into exosomes is based on their biological processes, cargo is loaded into donor cells or overexpressed certain gene products which are then packaged into exosomes , route 2MSC is the most well-suited for mass production of exosomes for drug delivery . For exaExogenous cargos can be directly loaded into exosomes by different methods see , and theEncouraging recent studies show exosomes as potential therapeutic vehicle systems for small molecule drugs to reduce toxicity and enhance target disease tissue in autoimmune diseases. Currently, GCs are the most effective anti-inflammatory drugs available for SLE , but cliAnother potential drug to load in exosomes is curcumin, which has immunomodulatory potential in addition to anti-oxidant and anti-inflammatory effects. In both acute and chronic immune nephritis curcumin ameliorated kidney disease reducing proteinuria, glomerulonephritis, tubule-interstitial disease and renal infiltration . In addiDelivering specific functional nucleic acid drugs to targeted cells by exosomes can regulate gene expression and maintain the physiological balance of the cells ,231. LiuExosomes also transported protein cargos, including shock proteins, major histocompatibility complexes, cytoskeletal proteins and proteins related to signal communication and membrane transport ,236. In In summary, these works illustrate the undeniable potential of exosomes as a therapeutic drug carrier in autoimmune diseases . NeverthMost autoimmune disorders such as SLE are characterized by a chronic inflammatory state, the making reduction of inflammation essential in order to treat patient conditions. Drug therapies for controlling SLE have made significant progress in recent decades. However, dosing limits of therapeutic indications in current SLE therapeutics delay their optimal use, and SLE patients may tolerate severe negative side effects produced by non-specific organ toxicity from frequent and long-term treatment. Compared with conventional therapeutic approaches, exosome-based drug delivery system has many advantages, such as high stability, compatibility, low immunogenicity and toxicity, longer circulation time and targeting ability for specific drug delivery to inflamed tissue. Therefore, exosome-based nanocarriers potentially have a bright future as next-generation drug delivery vehicles. This review has provided the current state of the art in the field of exosome-based drug delivery in SLE, evaluating the beneficial effects of natural and engineered exosomes on immunosuppression, cartilage repair and renoprotection.However, the clinical manufacturing of exosome-based therapeutics requires that comply with good manufacturing practice (GMP) to ensure reproducibility, stability and purity . TherefoDespite the challenges facing the exosome-based drug delivery system, these endogenous vesicles have great potential in the biomedical field. Furthermore, developing artificial exosome mimetics with similar homing properties and low side effects to exosomes could be an interesting concept to pursue to obtain clinical scale production of nanocarriers. In summary, the application of nanotechnology and nanomedicine provides great potential for prevention and treatment of human autoimmune diseases, especially in SLE therapy.


It is well established that breast cancer development and progression depend not only on tumor-cell intrinsic factors but also on its microenvironment and on the host characteristics. There is growing evidence that adipocytes play a role in breast cancer progression. This is supported by: (i) epidemiological studies reporting the association of obesity with a higher cancer risk and poor prognosis, (ii) recent studies demonstrating the existence of a cross-talk between breast cancer cells and adipocytes locally in the breast that leads to acquisition of an aggressive tumor phenotype, and (iii) evidence showing that cancer cachexia applies also to fat tissue and shares similarities with stromal-carcinoma metabolic synergy. This review summarizes the current knowledge on the epidemiological link between obesity and breast cancer and outlines the results of the tumor-adipocyte crosstalk. We also focus on systemic changes in body fat in patients with cachexia developed in the course of cancer. Moreover, we discuss and compare adipocyte alterations in the three pathological conditions and the mechanisms through which breast cancer progression is induced. In cancer, the tumor surrounding cells have altered biological properties compared with the normal state. We know today that there is a large spectrum of reciprocal interactions between a tumor and its stroma that significantly influence tumor biology and support tumor progression. Given the close juxtaposition of adipocytes and breast cancer (BC) cells, it is somehow surprising that knowledge-intensive research about the role of adipocytes in tumor initiation, growth, and metastasis is a relatively new area of investigation . The preFABP4) and the insulin-responsive glucose transporter type 4 (GLUT4) are characteristic of an early stage of differentiation  is greater than 25 or 30 kg/m2, respectively [According to the World Health Organization (WHO) and the National Institute of Health (NIH), overweight and obesity are clinically present when the body mass index [BMI, defined as weight (kg)/ height , confirming an 82% higher risk in post-menopausal women (95% CI = 55–114%) . The samSome meta-analyses analyzing large numbers of patients showed that obesity is also positively related to BC recurrence and mortality. Protani et al., evaluating 43 historical studies  and comparing obese versus non-obese patients, showed a statistically significant higher risk for overall survival (HR = 1.33) and for BC specific survival  in obese patients . In inteObviously, obese patients may have delayed diagnosis with more advanced disease at diagnosis compared with patients with normal BMI. This was shown by a study in Denmark on over 18,000 patients in which obese patients were older and had significantly more advanced BC at diagnosis than normal weight patients. However, after data adjustment for disease characteristics, obesity remained an independent prognostic factor for distant metastases and for death in BC . SimilarSeveral in vitro studies have demonstrated that AT supports and promotes tumor growth. Moreover, the degree of tumor infiltration into the adjacent fat AT serves as histological criterion reflecting the aggressiveness of the tumor and is indicative of poor prognosis ,59. A spNumerous studies demonstrated that adipocyte lipolysis stimulated by cancer cells is at the very heart of the synergy between cancer cells and adipocytes. Lipolysis is defined as the hydrolytic cleavage of ester bonds in triglycerides (TGs), resulting in the generation of fatty acids (FAs) and glycerol . BC cellThe pathological expansion of altered white AT in obesity leads to an abundant production of several biologically active factors, including inflammatory cytokines, hormones, adipokines, and lipid metabolites that can be considered mediators between obesity and cancer. Recent evidence demonstrated that CAAs have an altered secretome compared to mature adipocytes. CAAs share some common features with obese adipocytes, e.g., they secrete significantly higher levels of motility factors such as CCL2, CCL5, autotoxin (ATX), as well as proinflammatory cytokines such as IL-1β, IL-6, TNF-α, VEGF, and leptin. Moreover, cachectic adipocytes give an essential contribution to cachexia .It is well recognized that chronic inflammation with high circulating levels of C-reactive protein represents a pathophysiological condition that bridges obesity and cancer . HoweverIn normal AT, adipocytes are not the major source of IL-6, however, under pathological conditions such as obesity and cancer, the levels of IL-6 secreted from adipocytes increase significantly ,68. ChanTNF-α is an important inflammatory factor in the tumor microenvironment that is generated by tumor and stromal cells. Moreover, BC cells in coculture with adipocytes stimulated their TNF-α production . While, More than 75% of BCs express the ER, which means the vast majority of BCs grow and progress in response to estrogens. Of note, estradiol (E2), through the alteration of the tumor microenvironment, was shown to increase the growth of ER-negative BCs . EstrogeAdiponectin is the most abundant protein secreted by AT with wide range influence on several tissues and organs, performing insulin-sensitizing, anti-inflammatory, antiatherogenic, proapoptotic, and antiproliferative actions . Unlike Leptin is a hormone made by AT physiologically acting primarily on neurons in the hypothalamus, regulating food intake and energy expenditure. The role of leptin in tumorigenesis was suggested by the high expression of its receptor (ObR) in several cancer cells. Both leptin and ObR were found overexpressed in BCs, especially in higher grade tumors, and were shown to be related with distant metastasis and poor prognosis . PrevaleLeptin is a key paracrine mediator regulating the interaction between stromal cells and BC cells in the meaning of tumor metabolism. It was shown that leptin released by AT contributes to the metabolic features associated with BC malignancy, such as switching the cells’ energy balance from mitochondrial β-oxidation to the aerobic glycolytic pathway . ObesityAutotaxin (ATX) is a secreted glycoprotein produced by platelets, endothelial cells, fibroblast, adipocytes and, in varying degrees, by cancer cells . EnzymatResistin is a fat-derived secretory factor usually found in inflammatory zones . RecentlAdipocytes and preadipocytes are known to secret hepatocyte growth factor (HGF), which, together with its receptor present on tumor cells (c-Met), forms a signaling pathway intensely correlated with proliferation, metastasis, and angiogenesis . HGF wasInsulin-like growth factor 1 (IGF-1) is also secreted by adipocytes and preadipocytes, and its release is increased about two-fold in both undifferentiated cells and differentiated adipocytes from obese compared to lean individuals ,88. IGF-Cancer progression builds upon the ability of cancer cells to traverse the extracellular matrix (ECM) barrier access the circulation and establish distant metastases. The ECM composition not only orchestrates cancer and stromal cell behavior but also regulates the spectrum of reciprocal complex interactions between cells. It is widely recognized that both biochemical and biomechanical properties of the ECM influence cancer cell plasticity, allowing them to survive in hostile microenvironments and resist therapy. Production of ECM components functioning as signaling molecules and its continuous remodeling are clearly implicated in tumor progression. In AT, ECM is crucial for maintaining the structural integrity of adipocytes and plays a pivotal role in adipogenesis. Adipocytes are an abundant source of ECM components. However, tumor-induced adipocyte “activation” makes them produce even larger quantities of tumor promoting ECM components, such as collagen VI (COLVI) ,21, whicIn the same way, adipocytes at the invasive front of human tumors and CAAs in vitro exhibit increased expression of MMP11) ,38. MMP1Cancer cells invading regions of adipocytes in the tumor microenvironment interfere with essential functions of white AT, such as controlling energy balance, leading to the redistribution of nutrients in favor of cancer cells. It was shown that a tumor educates its stroma by downregulating p62, and such a prepared stroma shuts down energy utilization, which increases nutrient availability for cancer cells . RecentlTumor-educated adipocytes due to strong metabolic pressure shift their metabolism to glycolysis with concomitant release of energy-rich metabolites, such as lactate and pyruvate. The dynamic monocarboxylate shuttle between BC cells and adipocytes through monocarboxylate transporters (MCT) plays a role in BC aggressiveness . Indeed,Ketone bodies produced and released by glycolytic adipocytes are an ideal substrate for ATP production by driving oxidative mitochondrial metabolism in invasive cancer. They may burn more efficiently than other mitochondrial fuels, even during hypoxia, potentially allowing the tumor to grow in the absence of an optimal blood supply . ImportaCancer-associated cachexia is a well-known complication of cancer that frequently is the cause of death in cancer patients. As a systemic illness, cachexia affects the vast majority of patients with end stage cancer. Due to an energy imbalance condition and inflammation, the body burns its own components to sustain the tumor growth. Cachexia differs from malnutrition in as much as cachexia cannot be successfully treated by supplemental nutrition alone . CachexiHSL or ATGL) [Cachectic adipocytes under some aspects, such as morphologic modifications, decrease in lipid content , decreasor ATGL) , making or ATGL) ,157,158.A very reasonable question arises, namely, whether excess body fat and obesity can contribute to cancer cachexia occurrence and worsening patient prognosis. As i) illnesses associated with insulin resistance, such as obesity, exhibit increases in whole-body protein degradation, ii) insulin resistance is associated with muscle fat accumulation, and iii) high-fat diet enhances muscle protein catabolism associated with an increase in plasma FAs and a decrease in plasma adiponectin, the implication of obesity in cancer cachexia is an entirely plausible scenario . Body coTumor cells are highly energy-demanding, thus energy and metabolic intermediates such as FFAs released from AT in both obese and cachectic are desired substrates required to sustain their proliferation. However, certain cancers, along with high rate lipolysis, were shown to induce white-to-brown transdifferentiation, named “browning’’, mediated by UCP1, resulting in enhanced fat energy burning with concomitant fat tissue wasting . ParadoxThe process of cancer induced adipocyte browning is largely described in cancer cachexia and concerns adipocytes in the whole body, but this phenomenon has been also observed in the tumor microenvironment. Indeed, higher browning of adipocytes was noted in AT adjacent to BCs when compared to benign lesions . Highly While it seems possible that restoring AT homeostasis by targeting these areas of dysfunction would contribute to further improvements in patient-centered outcomes, current clinical trials investigating potential therapeutic agents for patients with cancer associated cachexia do not directly address the contributions of white, beige, and brown AT to cancer cachexia .Adipocytes are active players in the development and the progression of BCs. Through the secretion of several stimuli, they create a permissive microenvironment for tumor growth. In the current review, we compared adipocytes involved in three pathologies—obesity, BC, and cachexia. In our opinion, such a comparison may help to understand the way adipocytes support tumor growth and, above all, it can hopefully bring us closer to understanding how cancer cells introduce modifications in AT not found in normal AT. Adipocyte characteristics in obesity are similar to those detected in CAAs of invasive tumors, raising the possibility that adipocytes support tumor development and progression in obese people due to their already altered phenotype. At first glance, such different adipocytes observed in obesity and cachexia turned out to be very similar, especially in terms of metabolic disturbances and pro-inflammatory features, hence becoming dangerous partners of tumor progression. Importantly, altered phenotypes of CAAs and cachectic adipocytes are the results of tumor direct/indirect actions, local and systemic, respectively. Indeed, they both have detrimental effects when it comes to tumor progression and patient prognosis.Adipocytes, as active player in tumor progression, are emerging as a new target in treating BC. New drugs can be developed to target adipocytes and/or cancer cells to block the adipocyte–tumor cells’ vicious cycle from inducing cancer progression. In this context, further research is required to understand the mechanism(s) by which tumor cells modify adipocytes. Tumor-derived signals to adipocytes might be those used by tumor cells to continuously communicate with and activate stromal cells in the bone marrow or in distant organs in order to progress into metastasis. Moreover, blocking such signals could help in countering tumor-derived adipocyte dysfunction such as cachexia. On the other hand, understanding the efficacy of adipocyte-derived factor inhibition on cancer progression could represent a new strategy to treat cancer, especially in obese patients. Curiously, adipocytes have been also exploited to be the final result of a trans-differentiation process induced in BC cells to reduce BC progression . Indeed,


In coronary artery spasm (CAS), an excess coronary vasoconstriction causing total or subtotal vessel occlusion could lead to syncope, heart failure syndromes, arrhythmic syndromes, and myocardial ischemic syndromes including asymptomatic myocardial ischemia, stable and unstable angina, acute myocardial infarction, and sudden cardiac death. Although the clinical significance of CAS has been underrated because of the frequent absence of symptoms, affected patients appear to be at higher risk of syncope, serious arrhythmias, and sudden death than those with classic Heberden's angina pectoris. Therefore, a prompt diagnosis has important therapeutic implications, and is needed to avoid CAS-related complications. While a definitive diagnosis is based mainly on coronary angiography and provocative testing, clinical features may help guide decision-making. We perform a literature review to assess the past and current state of knowledge regarding the clinical features, electrocardiographic abnormalities and angiographic diagnosis of CAS, while a discussion of mechanisms is beyond the scope of this review. Angina pectoris is caused by a transient imbalance between myocardial demand and supply st article st reported variant form of angina pectoris, his description of non-exertional angina is vague. On the other hand, patients with Heberden's angina may also manifest CAS However, Dr. Myron Prinzmetal (1908-1987) published his observations on "A variant form of angina pectoris" in 1959, which was the 1st report of 32 cases of variant angina, of which 20 were personally observed and 12 were from the literature, the pain associated with transient non-progressive ST-segment elevation appeared at rest or during ordinary activity but was not brought on by exercise or emotional disturbance. Among the 32 patients studied, 12 developed myocardial infarction during follow-up st report of coronary angiography in 1959 In Prinzmetal's 1In the 1970s and 1980s, the diagnosis of CAS by coronary angiography in the catheterization laboratory was not rare. It then became clear that CAS could occur in patient with atherosclerotic obstructive coronary artery disease CAS-related angina and myocardial ischemia, regardless of symptoms, are common There are wide differences in the CAS prevalence in different countries. The frequency of CAS in Japan is greater than that in western countries Symptoms vary widely and may be silent Table , among wCAS occurs mostly at rest or at night, sometimes in association with awakening from sleep The incidence of CAS shows daily, weekly, monthly, and circadian variations CAS, especially multiple spasms In addition, the prevalence of atrial fibrillation in the patients with dilated cardiomyopathy and CAS is greater than that in dilated cardiomyopathy without CAS [67% vs 8% (P<0.05)] All CAS-related electrocardiographic changes may occur without the subsequent angina On the other hand, a normal electrocardiography does not rule out the presence of CAS The ST-segment and T wave changes of CAS are associated with the subsequent left ventricular dysfunction and practically every known form of arrhythmia Arrhythmias, particularly ventricular, appear more frequently through unknown mechanisms during CAS attacks in >50% of cases than during attacks of classic Heberden's angina pectoris On the other hand, severe CAS may cause fatal pulseless electrical activity or asystole without complications of ventricular tachycardia or VF For patients presenting with transient typical chest pain When ST-segment elevation appears, the diagnoses most often confused with CAS, which presents with transient non-progressive ST-segment elevation, include acute ST-elevation myocardial infarction, acute pericarditis, left ventricular hypertrophy, left bundle branch block, preexcitation, non-cardiac chest pain associated with early repolarization Although the key to the clinical diagnosis in medicine is the taking of a history, CAS may present with or without symptoms A recently developed clinical diagnostic score for prediction of CAS in patients with acute chest pain consists of 6 factors, including allergies, asthma, angina attack at rest, ST-segment elevation, myocardial bridge, and hyperventilation test st CAS, probably catheter- or contrast- related, with reproduced chest pain was demonstrated angiographically in a patient with Prinzmetal angina in 1972 st provocative testing using intravenous and intracoronary ergonovine were performed by the Cleveland Clinic in 1972 Coronary angiography with provocative testing, initially described in 1972 In patients with ST-segment elevation during episodes of angina and a normal coronary angiography, provocative tests usually are not necessary for diagnosis of CAS The diagnostic criteria of positive provocative testing for CAS was initially described by Chahine et al. according to The severity and location of CAS and the associated electrocardiographic abnormalities have been variable in different episodes of CAS During provocative tests, the occurrence of severe chest pain with ST-segment depression but without epicardial CAS has been termed microvascular CAS The provocative testing at present involves the use of ergonovine or acetylcholine To ensure a valid diagnosis, vasodilators  must be withdrawn for ≥24 hours except for sublingual nitroglycerin if necessary About one-third of patients with dilated cardiomyopathy has CAS and multiple episodes and locations of CAS may lead to HFrEF While the complications of intracoronary provocative testing include angina, hypotension, dyspnea, flushing, nausea, vomiting and various arrhythmias The management of angina should not be oversimplified as CAS not only induces angina but also causes HFrEF. Patients with HFrEF and CAS may have associated atrial fibrillation. In addition, asymptomatic CAS patients could die suddenly of VF, pulseless electrical activity or asystole. Awareness of CAS is important for clinicians for appropriate management, and preventing progression to more severe atrioventricular block necessitating permanent pacemaker implant. Furthermore, since the coronary vasoconstriction is nonspecific and the angina of CAS is not relieved by rest, CAS-related angina in the same patient may occur under different conditions. As a result, it is important to identify patients with CAS because treatment strategies are different between obstructive coronary artery disease and CAS, and the administration of calcium channel blockers is necessary to improve left ventricular function for CAS-relarted HFrEF.


Injectable biomaterials scaffolds play a pivotal role for dental tissue regeneration, as such materials are highly applicable in the dental field, particularly when compared to pre-formed scaffolds. The defects in the maxilla-oral area are normally small, confined and sometimes hard to access. This narrative review describes different types of biomaterials for dental tissue regeneration, and also discusses the potential use of nanofibers for dental tissues. Various studies suggest that tissue engineering approaches involving the use of injectable biomaterials have the potential of restoring not only dental tissue function but also their biological purposes. This is a narrative review, which discusses the state of the science of injectable biomaterials for dental tissue engineering from a theoretical and contextual point of view. Biomaterials, which can be either synthetic, natural and pure or composite materials play an important role in dental tissue engineering to interface with surgical procedures and cell biology ,2, and aInjectable scaffolds play an important role in tissue regeneration since they provide numerous advantages over pre-formed scaffolds  and are Hydrogels are broadly classified based upon their original sources, preparation method and physico-chemical behaviour, such as crosslinked, interpenetrating network, chemical and stimuli responsive and protein-based hydrogels, as depicted in Conventionally, based upon crosslinking, hydrogels are classified into two distinct categories: physically crosslinked and chemically crosslinked hydrogels .Hydrogels prepared by physically crosslinked methods are usually reversible gels and are relatively easy to produce without any crosslinking agents during synthesis process. The crosslinking agents may affect the integrity of components (cells/proteins) entrapped into the hydrogel system and need to be removed before hydrogel application. Moreover, hydrogels formed with non-covalent bonds may be moldable, permitting a better defect filling ability. This leads to an increased demand of physically crosslinked hydrogels in the field of biomaterials and tissue engineering .a)Radical polymerization: Swelling is an important characteristic of the hydrogel system and it can be regulated by the number of crosslinking agents incorporated into the system. Crosslinkers of predetermined properties result in the formation of stimuli-sensitive materials. Natural, synthetic and semi-synthetic polymers are considered to design hydrogels via this route .b)2 and COOH present in a polymer make it water soluble and can be modulated to develop a hydrogel. Establishment of covalent bonds between the polymer chains is generally achieved by functional groups and complementary reactivity interaction, such as, amine-carboxylic acid reaction by Schiff base formation.Chemical reaction of complementary groups: The functional groups OH, NHc)Ionic interaction: Crosslinked polymer systems generated by the inclusion of di or tri-valent counter ions falls in this category. The underlying principle is the gelling of polyelectrolytic solution with oppositely charged multivalent ions. For example, alginate—a polysaccharide with glucuronic and mannuronic acid residues can be crosslinked with calcium ions by ionic interactions at room temperature and physiological pH. Alginate hydrogels are used as templates to encapsulate living cells and release protein. The ionotropic hydrogels tend to degrade under physiological conditions .d)Crystallization: Aqueous solution of water-soluble polymer such as polyvinyl alcohol (PVA) when stored at room temperature forms stable gel. Interestingly, aqueous solution of PVA upon freeze-thawing tends to form a highly elastic and strong gel. The gel characteristics depend on molecular weight of PVA, concentration of the polymer in water, temperature, freezing time and number of freezing cycles .The chemically crosslinked hydrogels are stable elements as the covalent bonds exist in between the polymer chains. These hydrogels do not dissolve in any solvent until the covalent crosslinked points are weakened. The chemically crosslinked hydrogels are mechanically strong and depend upon the nature of chemical bonds existing between building blocks and crosslinks . Differea)Homo-polymeric hydrogels: Network of polymers derived from single monomer species consist of basic structural units. Homopolymers may possess crosslinked skeletal structure which depends upon monomer nature and polymerization technique. For example, polyethylene glycol (PEG) hydrogels are smart and drug delivery matrices due to their responsive nature to the external stimuli .b)Co-polymeric hydrogels: These hydrogels contain two or more dissimilar monomeric species, among which at least one is a single hydrophobic monomeric constituent assembled in block, randomly or alternating structural configuration along chain of polymeric network.c)Semi-interpenetrating network (semi-IPN): A linear polymer penetrating into another crosslinked network without chemical bonding is termed as semi-IPN. This network is capable of preserving rapid kinetic response rates with respect to pH or temperature due to lack of restricting interpenetrating network. The hydrogels possess modified pore size and exhibit slow release of drug. The network consists of both covalent and ionic bonds. The covalent bonds maintain three dimensional (3–D) structures of the hydrogels whereas the ionic bond imparts mechanical strength and pH responsive nature .d)Interpenetrating network (IPN): This is an intimate combination of two different kinds of polymers of which one is crosslinked or generated in immediate presence of other polymers. The interlocked structural property of crosslinked IPN system ensures stability and morphology of the hydrogels. These hydrogel templates have appreciable mechanical strength, controlled porosity, physico-chemical and mechanical properties leading to efficient drug loading .Based upon preparation method, hydrogels are classified as homo-polymers, co-polymers, semi-interpenetrating networks and interpenetrating networks and are described below:These hydrogels are environmentally sensitive and a smart hydrogel system as they experience unusual changes in the network structures, growth factors, permeability and mechanical properties in response to the environmental stimuli. Physical stimuli include light, temperature, pressure, magnetic and electric fields, mechanical stress and chemical stimuli such as pH, chemical agents and ionic factors, that are responsible in changing polymer chain/solvent interactions at the molecular level. The dual responsive hydrogel is a network of two stimuli responsive factors in a single hydrogel system and biochemical stimuli hydrogel responsive to ligand, enzyme and antigen have also shown potentialities in biomaterials field .a)pH responsive hydrogels: These hydrogels with ionic pendant groups response to environmental pH change and have the ability to accept or donate protons. This leads to volume transition to create osmotic swelling force by generating electrostatic repulsive forces in between the ionized groups. pH responsive hydrogels can further be classified as anionic hydrogels with pendant group of carboxylic or sulfonic acid and cationic hydrogels with pendant groups of amines. The degree of swelling of these hydrogels depend on many factors-concentration, crosslinking density, hydrophobicity or hydrophilicity, ionic charge and degree of ionization ,26.b)m) as positively thermo-sensitive hydrogels. The hydrogel swelling is reversible corresponding to step-by-step temperature change which has effect on release rate of model drug ketoprofen from monolithic device. Overall, the swelling response can be assessed in co-ordination with temperature variation to study the behaviour of hydrogels  and takes direct part in pulp capping procedure. One of the most important and promising application of EMD is to promote regeneration of functional PDL after avulsed teeth avulsion and replantation [Heijil et al.  demonstrantation .2 and MTA. Besides, EMD has been successfully used to treat large periradicular lesions of endodontic origin located adjacently to dental implants with regressive peri-implantitis [In terms of revascularization, Nakamura et al.  utilizedlantitis . Sarnastlantitis  found thPalioto et al.  evaluateSchwarz et al.  investigPEG is suitable for providing a temporary matrix . Galler Studies ,158,159 However, the use of these materials faces the challenge due to difficulties in insertion or in the modification to fit into narrow spaces of the pulp. As a result, there is the need for other materials which are simple to insert and to modify ,160. CurIn their study, Galler et al.,  tested dEndodontics procedure is defined as a biologically based system designed to replace defected dentin, root structure and complex pulp-dentin cells. During the development of an embryonic tooth, a cranial neural crest develops dental epithelium and ectomesenchymal cells originating from dental epithelium to give rise to dental papilla. During embryogenesis, differentiated ectomesenchymal cell aggregation or odontoblasts secrete primary dentin surrounding the dental pulp. The dental pulp is soft tissue placed in tooth core containing nerve and blood vessels subjected to chemical, thermal, mechanical and bacterial insults. Generally, infection in the pulp chamber due to bacterial infection may cause serious damage including tooth fracture and failed root canal treatment (RCT) .Endodontic tissue regeneration is based on the principle of conventional regenerative medicine. The regenerative endodontics involves research concerned with the use of growth factors, stem cells, organ tissue culture as well as tissue engineered matrix. Generally, highly structured mechanically strong 3-D scaffolds provide excellent support to the cells used in any area of body including bone where a tailored scaffold is needed to provide physical support. However, in case of root canal treatment or more specifically for pulp complex regeneration, strong scaffold cannot be placed to deliver structural support to the tooth. Thus, engineered pulp tissue is administered as 3-D soft templates such as polymer hydrogel.Injectable hydrogels are considered as outstanding biomaterials for therapeutic cell and bioactive molecules delivery for guided dental tissue engineering, especially for pulp complex regeneration applications. Hydrogels possess tuneable tissue-like characteristics, controlled degradation and release features, easy adaptability in clinical settings for minimal invasive surgery and shape conformation ability, i.e., to conform 3-D defects upon gelling. Endodontic tissue engineering is based on the principle of conventional regenerative medicine. In regenerative endodontics, injectable hydrogels have shown feasibility of delivering dental pulp stem cells, supporting template-like enamel derivatives and growth factors  to support dentin pulp complex formation. Controlled release of growth factors from dentin is an important defence mechanism against dental injuries . These gHydrogels made of natural polymers are highly biocompatible, inexpensive, readily available and easy to tailor by applying simple and cost-effective techniques. They have been investigated as injectable scaffolds worldwide for dentin-pulp complex regeneration. Some of the natural polymers include chitosan, collagen, gelatine, alginate and hyaluronic acid. The applications of these polymers in pulp regeneration are described briefly below.Feng at al.  demonstrOn the other hand, synthetic hydrogels possess finely tuned characteristics—biodegradability and high reproducibility with little batch to batch variation. The commonly used synthetic hydrogels for dental pulp regeneration: polylactic acid (PLA) and polyglycolic acid are described briefly. Scaffolds made of these polymers are conductive for seeding stem cells such as SHED and DPSCs. Stem cells were found to differentiate into odontoblast-like cells on these hydrogels, leading to endothelial cell stimulation to generate tissues resembling dental pulp and dentin. Mooney at al.  demonstrCordeiro et al.  investigPlatelet rich fibrin (PRF) is a fibrin matrix in which the platelet cytokines and cells are wedged to serve as a resorbable matrix following their release after a stipulated time. PRF was used initially in oral implantology, but, it has also shown wide application in pulp regeneration. PRF is considered as an appropriate scaffold material and a promising tool for regenerative endodontics due to its ability to control differentiation of stem cells and deliver growth factors in sustained and controlled manner. Platelet rich plasma (PRP)/PRF combined with collagen membranes was used extensively for periodontal ligament and alveolar bone regeneration as guided tissue in periodontics and oral surgery .Yang et al.  demonstrShivashankar et al.  reportedPhotopolymerizable and the biodegradable materials are getting popular with extended use and applications in relation to the field of tissue engineering such as bone, teeth, liver, and cartilages. The technology of photo-initiated polymer making has allowed the creation of injectable photopolymerizable biomaterials that is for dental and other tissue engineering applications . As suchVarious scaffolds such as polymeric scaffolds, natural polymers, synthetic polymer, self-assembling materials, hydrogels, extra-cellular matrix, and the bioceramics materials are used in the dental field as the materials for pulp tissue regeneration ,186,187.Scaffolds can be used in dental pulp regeneration. Moreover, combining stem cells with the inductive scaffold materials will optimize the regeneration of dentine pulp . The useNojoomi et al.  found thCalcium phosphate cements (CPCs) are used as injectable scaffolds for dental tissue regeneration engineering ,193. SimIn another study, Thein-Han et al.  examinedThe polymer chains present in hydrogels are crosslinked with each other either physically or chemically. Minute environmental condition changes such as pH, temperature, presence of enzyme or ionic species, electric signal might elicit fast and reversible changes in the hydrogel system. These changes usually take place at microscopic level as change in conformation, precipitation and water content of hydrogel . Few facThe swelling kinetics of hydrogels are classified as Fickian (diffusion controlled) and non-Fickian (relaxation controlled) swelling kinetics. The swelling kinetics is practically diffusion controlled when water diffusion into hydrogels occurs at faster rate than polymer chain relaxation. The crosslinking ratio is one of the best factors to influence swelling of hydrogels. The higher the crosslinking ratio, the more the crosslinking agent is interlinked in the hydrogel network. The highly crosslinked hydrogel system has a more organized and compact structure and tends to swell in small amounts as compared to hydrogels with low or without crosslinking ratios. Generally, crosslinking hinders the polymer chain mobility, thus, the swelling ratio is decreased. The chemical structure of hydrogel also influences its swelling ratio .The mechanical strength of hydrogel is increased with the increase in crosslinking density, leading to the formation of stronger and stiffer gels. The increase in crosslinking density also results in lowering of percentage (%) elongation of the hydrogel. Hence, the hydrogel turns brittle in nature. Many researchers have demonstrated co-polymerization with H-bonding increment within the hydrogel system to attain desirable mechanical properties.Synthetic hydrogel polymerization contains toxic elements derived from organic solvents, emulsifiers, and stabilizer. Mostly, crosslinkers present immense challenges for in vivo biocompatibility. Natural polymers are considered to have better biocompatibility over synthetic polymer-based hydrogels. However, natural polymers incorporated with synthetic crosslinkers or initiators for polymerization possess similar toxicity concerns such as that of the synthetic hydrogels .The electrospinning technique is one of the versatile techniques, which is very popular because of its broad applications in the dental and medical areas. The electrospinning technique is focused on its applications in tooth regeneration, healing and preventing diseases such as dental caries. The materials used with the electrospun technique possess unique characteristics such as a higher surface area to volume ratios, have improved cellular interactions, and better protein absorption, and thus the enhanced absorption that leads to good binding sites and enhanced cells reception ,205. OthCompared to collagen nanofibrous scaffolds made through integrin-intermediated procedure, the use of polycaprolactone (PCL) and collagen-based nanofibrous scaffolds made via electrospinning integrating bioactive glass nanoparticles to regenerate dentin-pulps enhances odontogenic and growth differentiation from human dental pulp stem cells (hDPSCs) ,210,211.Electrospun nanofibers such as polymeric nanofibers or bioceramic nanoparticle-integrated nanofibers are applied in dentistry. In addition, their flexibility and nanostructure have resulted in highly promotive cell homing behaviours, leading to enhanced dental health . In addiKuchler-Bopp et al.  examinedAurrekoetxea et al.  found thNon-toxic, biocompatible and can undergo biodegradation;Allows biofunctionality of cells such as odontoblasts, ameloblasts, fibroblasts, cementoblasts, vascular cells and neural endings;Fabrication of natural or synthetic polymers or combination of both as hydrogels (preferably injectable hydrogels);Reasonable shelf life, can be sterilized and stored in clinical settings and key approach for clinicians.Biomaterials are mostly indispensable for dental application, mainly for periodontitis treatment, endodontic pulp-dentin complex reconstruction and overall tooth regeneration. Soft biomaterials such as hydrogels may have the potentialities to serve as cell encapsulating matrix. However, a tooth should assume appropriate anatomical dimension and shape to function in the dentition . An apprGenerally, cell doctrine, scaffolds and biological signals are the principle guiding elements for tissue engineering. The bioactive scaffolds consist of embedded bioactive molecules/cells to serve the purpose. Transplantation and homing of cells are the scientific approaches of tissue regeneration. The biomolecules such as proteins, peptides and RNA/DNA can be infused in vivo to establish new environment to promote dental regeneration.On the other hand, cell free approaches utilizing bioactive scaffolds, cells as well as growth factors from dentin behave like chemoattractants for endogenous stem cells that reside in the periapical region or in the surrounding healthy pulp tissue. These cells are expected to adhere, proliferate and differentiate to give rise to new tissue, and thus the physiological tissue condition of dental pulp is mimicked. Periodontal regeneration remains a seriously challenging task as the existing regeneration techniques have not achieved predictable periodontal tissue regeneration in humans. The synergistic combination of tissue engineering and nanomedicine has led to a remarkable progress in the periodontal tissue engineering field. Several polymers such as chitosan, collagen, alginate and hyaluronic acid as hydrogels have been investigated to promote successful guided tissue regeneration . One of