This is a focused review discussing quality assurance during interventional brachytherapy in gynecological cancers. This topic is very large and is usually addressed from the technical and physical sides, therefore, we decided to select “hot-spots” under this large title and discuss them from the point of view of clinicians. We hope that this concise and focused review will help clinicians in improving their quality assurance protocols and draw attention to the discussed issues.The use of brachytherapy  in the treatment of gynecological cancers is a crucial element in both definitive and adjuvant settings. The recent developments in high-dose rate remote afterloaders, modern applicators, treatment-planning software, image guidance, and dose monitoring systems have led to improvement in the local control rates and in some cases improved the survival rates. The development of these highly advanced and complicated treatment modalities has been accompanied by challenges, which have made the existence of quality assurance protocols a must to ensure the integrity of the treatment process. Quality assurance aims at standardizing the technical and clinical procedures involved in the treatment of patients, which could eventually decrease the source of uncertainties whether technical (source/equipment related) or clinical. This commentary review sheds light  on some potential sources of uncertainties associated with the use of modern brachytherapy in the treatment of gynecological cancers. Image-guided adaptive brachytherapy (IGABT) is a crucial component in the treatment of different gynecological tumors. Despite the major recent technical advances in external beam radiation treatment (EBRT), IGABT is still superior to intensity modulated radiation treatment (IMRT) using photons or protons in terms of target coverage as well as sparing of the organ at risk (OAR) . Dose esQuality assurance (QA) for IGABT aims at standardizing the technical and clinical procedures involved in the treatment of patients, which could eventually decrease the source of uncertainties, both technical, such as source calibration, and clinical, such as delineation. However, dose planning and delivery are always related with the technical and physical properties of brachytherapy afterloaders ,11,12,13Furthermore, QA also guarantees the reliability of the evaluation process of the treatment results, in terms of local control and treatment-related toxicity. Treatment results are usually correlated to dose parameters for the target and the OAR as obtained from the dose volume histogram (DVH), which is an important indicator for setting the target prescription dose as well as the dose limits for the OAR.A crucial element in the QA of IGABT is the proper selection of applicators; all clinical and technical factors should be considered before a decision is made on the use of a certain applicator for each individual patient. Appropriate applicator selection is fundamental for tumor dose coverage, which is directly linked to rates of local disease control . A propeInappropriate applicator selection or inappropriate placement results in a poor tumor coverage which could not be compensated for by optimization, resulting in lower local disease control and lower disease-free survival. Furthermore, attempts to obtain better coverage through optimization may even result in dangerous hot spots and end in increasing toxicities ,17.Brachytherapy for gynecological tumors could be implemented through an intracavitary, interstitial, or hybrid (combination) procedure. In the case of vaginal-vault adjuvant brachytherapy or non-operable uterine cancer, an intracavitary application is usually selected, as is often the case also for small primary vaginal tumors. However, in large, deeply infiltrating tumors, or when involving the vulva or perineal tissue, a combined intracavitary and interstitial approach might be the most appropriate .The selection of a specific approach for IGABT in cervix tumors is highly dependent on the anatomy and extent of original as well as residual disease after EBRT (± chemotherapy) . The twoGynecologic IGABT implants require multidisciplinary and large-volume centers. As with to all surgical procedures, user-dependent skill and experience are key factors for their success ,23.MRI is superior in demarcating the extent of gynecological tumors and also in the delineation of the OAR, and consequently with its use better DVH value results are to be anticipated. However, although this seems an obvious advantage, studies have shown that the delineation of the OAR and doses did not differ significantly when comparing MRI and CT-based planning ,25. CT mPast studies have shown that high-risk clinical target volumes (HRCTVs) in cervix tumors delineated by CT (HRCTVct) are statistically different from MRI-delineated HRCTVs (HRCTVmri), even with pre-planning MRI ,27,28,29Due to the inferiority of CT in defining the limits of tumors, especially when preceded by EBRT (± chemotherapy) as in the treatment of cancers of the cervix, using CT usually leads to an overestimation of the tumor volume especially towards the rest of uterus cranially as well as along the parametrial extensions ,27,30,31The use of ultrasound (US)  in most centers is usually limited to guiding the placement of the applicator and possible interstitial needles, however combining three-dimensional abdominal or trans-rectal US images with CT has resulted in the better definition of tumor tissues and reduced overestimations of target volumes, creating treatment plans that were closer to MRI-based plans ,33. UsinImage registration is routinely used in the modern EBRT to evaluate setup errors and to assess the need to adapt the treatment along the course of treatment. In gynecological IGABT, image registration is used for applicator reconstruction, definition, or propagation of target volume, or for dose accumulation . Most raAccurate applicator reconstruction is a crucial factor in decreasing the uncertainties during IGABT, as for each 1 mm that an applicator is displaced during the reconstruction there is a 5–6% change in the mean DVH parameters . It is aAlthough MRI-based IGABT is superior in delineating target volumes and the OAR, it may be challenging for direct applicator reconstruction as the source path (applicator lumen) is difficult to identify especially on T2 images which are recommended for delineation ,47. It mIn hybrid implants, where interstitial needles are combined with rigid applicators, the accurate reconstruction of the tip of each needle on MRI may be highly challenging . Using tIn order to reduce uncertainties of contouring and maintain quality during IGABT, several factors are to be considered. High quality planning on thin-slice images enables the accurate delineation of the target as well as the OAR. If MRI is not used for planning, CT image registration with MRI and/or PET-CT improves the inter-observer contouring agreement ,52. GEC The GEC ESTRO has over the past years published recommendations on the delineation of target volumes in gynecological BT ,56,57. TThe use of both 3-D imaging and stepping sources enables dose prescription to be directed at a volume instead of fixed geometric points, and also enables a high level of plan optimization and dose-painting . In cancPlan optimization could start off automated and be followed by manual optimization, or could be performed purely manually. For cervical treatment, for example, usually, a standard point-A-based dose distribution (pear shaped) is performed, followed by progressive manual optimization through activation or inactivation of dwell positions and varying dwell times whilst monitoring the impacts of the treatment on spatial dose distribution as well as DVH parameters for both target volumes and the OAR .The GEC ESTRO recommended DVH parameters for target volumes and the OAR are widely accepted and endorsed. An important indicator for the covering of targets is the reporting of D100 and D90, which are the minimum doses covering 100% and 90% of the target volume, respectively . HoweverHigh-dose volumes are of high value as they may contribute to the resulting local control rates. Heterogenicity in the dose distribution as well as steep absorbed-dose gradients are typical qualities of brachytherapy, therefore a considerable volume of the target received doses as high as 200% of the prescribed dose. After considering this unique feature of brachytherapy as well as the fraction size and the absorbed dose-rate, the calculation of the biologically effective dose across the target becomes also heterogenous reaching its maximum in the target sub-volumes located near the applicator and/or the interstitial needles. In order to evaluate these high-dose volumes, the ICRU report 89 recommended reporting of D50 .In-vivo dosimetry (IVD) in gynecological brachytherapy has been typically used for dose verification , howeverA QA protocol regulates all the administrative procedures, technical aspects, treatment steps, and audit measures to ensure a consistent and safe fulfilment of the treatment prescription. A qualified medical physicist should be responsible for the QA protocol regarding the appropriate description, calibration, and the current source strength . HoweverThe European Society of Therapeutic Radiology and Oncology (ESTRO) has published a booklet on quality control guidelines for brachytherapy . LikewisConsidering the available international literature and national regulations, as well as institutional resources and needs, medical physicists, radiation oncologists, radiation technologists, and all members of a brachytherapy team should work together to build an institutional QA protocol to standardize and document the clinical workflow.Gynecological brachytherapy has been significantly transformed from the era of 2-D to the era of 3-D and IGABT, with this huge development being led by major technological advances over the past years. Modern IGABT is a valuable advanced and complex tool in the treatment of gynecological tumors; its proper use requires dedicated training and a solid QA protocol must cover not only the physical and technical aspects of the treatment but also the many clinical considerations in order to decrease all possible sources of uncertainties and thus improve the treatment outcome and reduce toxicity.


Heart failure is the leading cause of death worldwide, and the most common cause of heart failure is ventricular dysfunction. It is well known that the ventricles are anisotropic and viscoelastic tissues and their mechanical properties change in diseased states. The tissue mechanical behavior is an important determinant of the function of ventricles. The aim of this paper is to review the current understanding of the biomechanics of ventricular tissues as well as the clinical significance. We present the common methods of the mechanical measurement of ventricles, the known ventricular mechanical properties including the viscoelasticity of the tissue, the existing computational models, and the clinical relevance of the ventricular mechanical properties. Lastly, we suggest some future research directions to elucidate the roles of the ventricular biomechanics in the ventricular dysfunction to inspire new therapies for heart failure patients. Despite the advances in modern management, heart failure (HF) leads to high mortality and morbidity in the United States. More than 5 million Americans have HF, and around 550,000 new cases occur every year ,2. It isVentricle dysfunction is the most common cause of heart failure, including left-sided HF with preserved ejection fraction (HFpEF) and reduced ejection fraction (HFrEF), as well as right-sided HF secondary to pulmonary hypertension and congenital heart disease (CHD) ,12,13,14It is generally accepted that the mechanical property of the myocardium is an important determinant of the ventricular function ,20. IndeThe ventricular free wall is known as an anisotropic and viscoelastic material, which means it has different mechanical behaviors in different directions and presents both elastic and viscous features in dynamic deformations . DependiAs needed in other biological tissues’ mechanical tests, preconditioning is often performed prior to the data acquisition to ensure a constant and accurate mechanical behavior of the tissue . This prResidual stress is the stress that remains in the tissue after all external loads are removed . The preUniaxial and biaxial mechanical tests are the most common methods to investigate the ventricular mechanical property after tissue harvest . While tFurthermore, when the entire cycle of stress–strain data is used , the ventricular viscoelastic behavior can be derived from the hysteresis stress–strain loop . HoweverStress relaxation and creep tests are traditional methods to measure the viscoelasticity of soft tissues such as tendon, cartilage, and heart valves ,51,52,53The ex vivo measurement discussed above can provide better controls of the experimental conditions  and eliminate the interference of physiological factors  in the mechanical properties of the ventricle tissues. However, the ex vivo tests require tissue removal and are often limited by the contractile state of cardiomyocytes (passive only) and the configuration of the tissue . Therefore, the in vivo measurements could provide useful information of the tissue mechanical behavior that is absent in the ex vivo conditions.systolic stiffness of the ventricle [At the whole-organ level, pressure–volume (PV) loop measurement  is the gentricle ,60.Non-invasively, cardiovascular magnetic resonance (CMR) and speckle-tracking echocardiography (STE) are alternative methods to measure the myocardial performance ,66,67. DThe direct non-invasive measurement of ventricular stiffness  can be obtained by magnetic resonance elastography (MRE) ,76. MRE Finally, with the combination of medical imaging and computational modeling such as finite element methods, it is also possible to estimate the ventricular material properties using ‘inverse modeling’ ,80,81,82The viscoelasticity of ventricles has been occasionally reported with the measurement of cyclic stress–strain relations. Some early studies measured the viscoelasticity of the LV from healthy canine and human hearts by individual measurements of pressure and volume in vivo ,87,88. BThe characterization of the anisotropic behavior of ventricles is highly dependent on the definition of the biaxial coordinate system. To date, there are two main types of coordinate systems: the main fiber and cross-fiber coordinate system ,30,44, aUsing the second coordinate system, Valdez-Jasso et al. found that the rat RV had greater stiffness in the outflow tract direction compared with the cross-outflow tract direction , and HilThe viscoelastic property of a material is manifested by the non-overlapping of loading and unloading stress–strain curves over an entire cycle . Such beBoth empirical models and constitutive models have been applied to characterize the nonlinear, biaxial mechanical behavior of ventricles. Because of the nonlinear, ‘J’-shaped stress–strain curve, the use of an exponential component is common in empirical models. However, these models provide little information on the relations of physical quantities or physiological conditions of the tissue, and thus constitutive models are developed to better describe the myocardium tissue mechanics . With cenon-cardiovascular tissues [Classic empirical models to describe the tissue viscoelasticity are composed of springs and dashpots that represent the elastic and viscous behaviors, respectively. The two basic models of these are also known as the Maxwell model (consisting of a spring and a dashpot in series) and Kelvin–Voight model . Different combinations of the spring and dashpot elements have been used to describe the ventricle and papillary muscle viscoelasticity. For example, a spring connected to two Maxwell elements in parallel was used to form a 1D viscoelastic model for the papillary muscle of the LV . An elas tissues ,94,95,96 tissues ,97,98.Heart failure is associated with extensive remodeling of the tissue involving changes in extracellular matrix (ECM) , recruitment of inflammatory cells , upregulated oxidative stress , and altered metabolic activity  ,101,102.The myocardium ECM consists of proteins such as collagen, elastin, fibronectin, proteoglycan, and laminin. Among these molecules, collagen is the most abundant ECM protein in the adult heart, with at least five different types of collagen  that have been identified . Types IVentricular fibrosis  is frequently observed in cardiac remodeling in both LV failure and RV failure ,108,109,While these previous studies investigated the role of fibrosis in the HF progression, the link of collagen deposition to the mechanical changes is another open area of research. Some biomechanical studies have quantified both biaxial mechanical properties and collagen/myo-fiber orientation in the ventricle  ,29,45. HIn chronic heart diseases, the myocardial structure and morphology changes lead to the stiffening of the ventricles ,120,121.In addition, the increased passive stiffness could result in an increase in stiffness during the systolic contraction, which is why the age-related increases in Ees  and EDPVR are correlated, regardless of the changes in arterial load . While EFinally, the stiffening of LV could impact on the pulmonary circulation as well. Pulmonary edema and elevation in pulmonary venous pressures are observed as a result of the backward transmission of elevated left-sided pressures into the pulmonary circulation. This leads to the development of post-capillary pulmonary hypertension (PH), which is commonly found in HFpEF patients . TherefoRV stiffening is consistently observed in a variety of PH etiologies as well as left-sided heart failure. Using non-invasive echocardiography, reduced RV longitudinal strains have been reported in pre-capillary PH  patients and PH patients with other etiologies ,129,130.The viscoelastic properties of the ventricle can impact the in vivo function. To date, the discussion of the relevance of ventricular viscoelasticity is mainly restricted to the diastolic function. Firstly, because the viscoelastic property is strain-rate dependent and because the early and later diastole have different filling rates, the diastolic function of the ventricle is time-dependent ,132,133.It is well accepted that the passive mechanical properties of the ventricle are important for the diastolic function, and thus heart diseases with a change in myocardial mechanical properties are often associated with diastolic dysfunction ,137,138.


Ferroptosis is a recently identified nonapoptotic form of cell death whose major markers are iron dependence and accumulation of lipid reactive oxygen species, accompanied by morphological changes such as shrunken mitochondria and increased membrane density. It appears to contribute to the death of tumors, ischemia-reperfusion, acute renal failure, and nervous system diseases, among others. The generative mechanism of ferroptosis includes iron overloading, lipid peroxidation, and downstream execution, while the regulatory mechanism involves the glutathione/glutathione peroxidase 4 pathway, as well as the mevalonate pathway and the transsulfuration pathway. In-depth research has continuously developed and enriched knowledge on the mechanism by which ferroptosis occurs. In recent years, reports of the noninterchangeable role played by selenium in glutathione peroxidase 4 and its function in suppressing ferroptosis and the discovery of ferroptosis suppressor protein 1, identified as a ferroptosis resistance factor parallel to the glutathione peroxidase 4 pathway, have expanded and deepened our understanding of the mechanism by which ferroptosis works. Ferroptosis has been reported in spinal cord injury animal model experiments, and the inhibition of ferroptosis could promote the recovery of neurological function. Here, we review the latest studies on mechanism by which ferroptosis occurs, focusing on the ferroptosis execution and the contents related to selenium and ferroptosis suppressor protein 1. In addition, we summarize the current research status of ferroptosis in spinal cord injury. The aim of this review is to better understand the mechanisms by which ferroptosis occurs and its role in the pathophysiological process of spinal cord injury, so as to provide a new idea and frame of reference for further exploration. As an intrinsic phenomenon of metabolism, cell death serves as an exploratory topic in the cryobiology sector, such as embryonic development, homeostasis, neoplasia, and tissue renewing. Previous scholars have found there are different forms of cell death, including accidental cell death (ACD) and regulated cell death (RCD) , 2.Among them, ACD is caused directly by physical, biological, or chemical factors that cause irresistible and irreversible damage to the plasma membrane or other components of the cell, such as organelles, rendering the cell unregulated and causing death. This type of death is often accompanied by the destruction of the cell membrane structure and obvious inflammation. ACD cannot be regulated by cells, so it is also called unregulated cell death . RCD, alAs early as 1956, scholars put forward the concept of PCD, which refers to a process of active death strictly regulated by cell signals, involving a series of gene activation, expression, and regulation. In addition to the earliest known apoptosis , 8, ferrFerroptosis is a form of cell death marked by the requirement for iron and the accumulation of ROS. Early research on ferroptosis mainly focused on cancer , 17, 18,An essential nutrient for the human body, iron plays an active role in maintaining physiological functions. It not only participates in the composition of heme in hemoglobin but also serves as a coenzyme in various catalytic reactions. There are two states of iron in the human body, bound iron, and free iron. Bound iron exists mainly in the form of ferritin, such as hemoglobin and iron-sulfur nanoclusters, while free iron, also called nonbinding iron, mainly exists in heme or non-iron-sulfur nanoclusters. Free iron includes both ferrous iron and ferric iron. Ferrous iron has high reactivity and increases the cytotoxicity of ROS.The proper level of iron maintains normal physiological function of the human body, but excessive free iron will cause harm to cells. As the donor of intracellular electrons, free iron undergoes redox transformation between ferrous iron and ferric iron. Ferrous iron generates (OH) hydroxyl radicals by means of the Fenton reaction, then induces oxidative stress and causes cell damage.Ferroptosis was originally postulated in 2012 by Stockwell and Dixon, American scholars. They discovered the inhibiting effects of Erastin and RSL3 on human RAS mutation fibrosarcoma cell line HT-1080 and proved it was the result of ferroptosis. Different from the canonical cell death pathway , ferroptosis depends on intracellular iron and has morphological, biochemical, and genetic features . MorpholIn essence, whether ferroptosis occurs is dependent on the balance between the ROS induced by iron enrichment and the antioxidant system that prevents lipid peroxidation. Once imbalance occurs, such as through excessive ROS production or decreased activity of the antioxidant system, it can trigger lipid peroxidation to damage the plasma membrane and induce iron death . CertainFerroptosis is an iron-catalyzed lipid peroxidation process, the most notable feature of which is the formation of ROS, which can be induced by a nonenzymatic mechanism (Fenton reaction) or enzymatic mechanism (lipoxygenase) . The mai3+ in the blood forms a complex with extracellular transferrin (Tf), which binds on transferrin receptor 1 (TFR1) on the cell membrane and transplants into the cell in the process of endocytosis Sec), which was attributed to the same UGA of the Sec genetic codon and stop codon. The translation process aided by tRNA[Ser]Sec can avoid the termination codon expression, so that it specifically translates Sec. tRNA[Ser]Sec regulates the Sec translation pathway, thus directly affecting the efficiency of the translation. IPP, the metabolic intermediate generated by the MVA pathway [[Ser]Sec and ensure the maximized efficiency of the UGA codon translation. Mature tRNA[Ser]Sec can specifically translate Sec and integrate the group into the active center of GPX4 to guarantee its activity.The in-depth understanding of the transcription and translation mechanism of selenoprotein has enhanced the study of GPX4 expression and activation regulation. Early studies showed that the active center of selenoprotein GPX4 was Sec, and its integration into GPX4 required the transport of specific mature tRNA , whose selenium atoms in the GPX4 protein of the body were all replaced by sulfur. Results showed the contents of parvalbumin-positive neurons were greatly decreased, and all the selenium-deficient mice died of fatal epilepsy by 18 days. Additional studies revealed GPX4 is critical to the maturation of mouse parvalbumin-positive neurons after birth. When selenium in GPX4 is replaced by sulfur, the activity of GPX4 decreases significantly and the accumulation of peroxides in the parvalbumin-positive neurons cannot be effectively removed, resulting in the death of a large number of neurons and eventually leading to severe epilepsy and mouse death [In 2018, 200 years after the discovery of selenium, Ingold et al.  found thse death .Research published in 2019 showed  seleniumSCI is an acute traumatic disease of the central nervous system, which always leads to different levels of motor, sensory, and autonomic dysfunction, and thus reduces the activity of daily living and the quality of life. The mortality and disability rate of SCI is high, and a sizeable proportion of patients has permanent dysfunction and cannot take care of themselves. As rehabilitation therapy is restricted and its curative effect is unsatisfactory, treatment of SCI is one of the medical problems that have troubled mankind. The reason why SCI is difficult to treat is not only the irreversible characteristics of nerve injury but also the mechanism of SCI which is a complex process which can be divided into primary injury and secondary injury. Primary injury refers to mechanical contusion or extrusion occurring at the moment of injury, which depends on the twisting force, compression force, and nerve transection degree, and is often irreversible. Secondary injury is the continuation and development of the primary injury, which is a complex cascade amplification reaction involving multiple mechanisms, including spinal cord tissue edema, oxygen free radical injury, inflammatory reaction, calcium overload, ion channel damage, glutamate toxicity, mitochondrial dysfunction, blood spinal cord barrier destruction, axonal demyelination, necrosis, and apoptosis, and its harm even exceeds that of the primary injury . Due to Does ferroptosis exist in SCI? Some scholars have found that the ferroptosis markers in the spinal cord tissue of SCI rats were obviously changed, and using transmission electron microscopy, they also observed changes in the mitochondria characteristic of ferroptosis, thus confirming that ferroptosis plays an important role in SCI . After SSubsequent studies confirmed that ferroptosis was an important cause of the serious consequences of secondary injury after SCI, and DFO could promote the recovery of motor function in SCI rats by inhibiting ferroptosis . Galluzz10/DADH pathway is considered to be a parallel and independent ferroptosis inhibiting pathway to the GSH/GPX4 pathway, and what is the distinctive mechanism of this pathway? How can FSP1 enter the cell membrane and play the role of reducing CoQ10 and how can CoQ10-H2 inhibit lipid peroxidation are still unknown. Selenium intervention in ferroptosis remains in the animal experimental stage and far from clinical application.Although considerable progress has been made in the study of ferroptosis, there are still many problems to be solved. For instance, what is the detailed mechanism of iron metabolism starting and subsequent oxidation reactions? The answer may help to prevent secondary injury caused by ferroptosis in SCI from the source. The newly discovered FSP1/CoQforms of cell death deserve our further exploration.Previous studies have confirmed that apoptosis after SCI is an important cause of neuronal death . AutophaAs a newly discovered form of cell death, ferroptosis has attracted a lot of attention in recent years and research on it has made immense progress. The mechanisms of ferroptosis include iron metabolism, lipid peroxidation, ROS accumulation, the GPX4 regulatory pathway, the MVA pathway, Se regulation, and the FSP1-mediated pathway. However, research concerning ferroptosis in SCI is still in its initial stage, and there remain a number of problems to be studied.


In recent years, polyetheretherketone (PEEK) has been increasingly employed as an implant material in clinical applications. Although PEEK is biocompatible, chemically stable, and radiolucent and has an elastic modulus similar to that of natural bone, it suffers from poor integration with surrounding bone tissue after implantation. To improve the bioactivity of PEEK, numerous strategies for functionalizing the PEEK surface and changing the PEEK structure have been proposed. Inspired by the components, structure, and function of bone tissue, this review discusses strategies to enhance the biocompatibility of PEEK implants and provides direction for fabricating multifunctional implants in the future. Bone defects caused by aging, trauma, disease, congenital abnormalities, and surgical resections are widespread all over the world. It's reported that the number of fractures is estimated to rise from 2.1 million to over 3 million during 2005–2025 in the United States  family, was first synthesized by British scientists via nucleophilic displacement in 1972  , which is the primary inorganic constituent of human bone, has been extensively studied in bone tissue engineering owing to its superb biocompatibility, osseointegration, osteoconduction, and osteoinductivity 6 (OH)2, in which the Ca/P ratio is approximately 1.67  is widely used in bone regeneration since its composition is close to that of natural bone  can also be deposited onto the surface of PEEK using several coating methods. Before sol-gel coating, pretreatment with UV, O2 plasma, or sandblasting can enhance the bonding strength of the coating layer, while post-treatment with HCl can induce apatite formation, which is related to the bone-bonding ability in vivo , which is one of the strongest osteoinductive factors, can initiate the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and promote osteogenesis , transforming growth factor-β, placental growth factor, and fibroblast growth factor can regulate angiogenesis, which involves endothelial cell proliferation, migration, and tube formation  proteins  and benefit the adhesion of osteoblasts  of PEEK implants have been used to enhance MG-63 cell behavior and bone-bonding ability  to PEEK via powder mixing and compression molding demonstrated that the enhanced surface roughness effectively promoted cell attachment and new bone regeneration  and fused filament fabrication have been adopted to construct PEEK scaffolds with controlled pore size  of cortical bone endows it with stability and allows it to support the inner porous structure , and graphene nanosheets (GNS) can be introduced into the PEEK matrix to satisfy the requirements and broaden its applications in load-bearing areas  have been adopted to treat infections caused by implants  loaded in ceramic cements could inhibit the growth of osteosarcoma cell line  first-generation implants possessing outstanding mechanical properties; (2) second-generation implants with prominent cytocompatibility and osteogenic activity; and (3) third-generation implants equipped with outstanding osseointegration and anti-inflammation/-infection ability. According to previous studies, we have grouped PEEK modifications into four categories based on their inspiration: the constituents , structure (surface roughness and porosity), mechanical function, and immune function of human bone.in vivo and elucidate which strategies are appropriate to modify PEEK in clinical settings. In this respect, the complexity, reproducibility, stability and cost-effectiveness of the manufacturing process should also be considered. In the future, researchers will focus on PEEK implants possessing the optimal combination of osteointegration, vascularization, anti-inflammation/-infection, and mechanical properties. It is worth noting that modifications that mimic the components, structure, and function of human bone are expected to be the key to fabricating multifunctional implants. Also, it can provide inspirations for researchers to modify other PAEK materials and expand their applications in the field of orthopedic, spinal and dental impalnts.Although the techniques for modifying PEEK have matured, numerous remaining obstacles must be overcome before modified PEEK implants can be applied in clinical practice: (1) the ratio of different constituents must be optimized to maximize bone regeneration and the mechanical properties; (2) methods must be developed to control the release of biomolecules and achieve the desired long-term effects; and (3) experiments are needed to identify the ideal pore structure to optimize biological performance. Thus, extensive studies are still needed to observe the long-term implantation of PEEK XG conceptualized and designed the manuscript. XG and XS drafted the manuscript. YS and JW designed the table. YL, KY, and YW designed the figure. YZ revised the manuscript. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YZ declared a shared affiliation, with no collaboration, with the authors to the handling Editor at the time of the review.


Biofilms are formed by the congregation of one or more types of microorganisms that can grow on a firm surface. Dental plaque is one of the most commonly forming biofilms in the oral cavity and appears as a slimy layer on the surface of the teeth. In general, the formation is slow, but biofilms are very adaptive to the changing environment, and a mature biofilm can cause many health-related problems in humans. These biofilms remain unaffected by antibiotics as they do not allow the penetration of antibiotics. Moreover, the increased level of virulence and antibiotic resistance of microorganisms in the oral biofilm or dental plaque has made its clinical management a serious challenge worldwide. Chlorhexidine-like antimicrobial drugs have been partially effective in removing such organisms; however, the precise and continuous elimination of these microorganisms without disturbing the normal microbial flora of the oral cavity is still a challenge. This review paper focuses on the process of oral biofilm formation, related complications, development of drug-resistant bacteria in these biofilms, and their effective management by the use of different novel techniques, available from various published research and review articles. Biofilm can be defined as a layer of both Gram-positive and Gram-negative bacteria, which adhere and grow on any biotic or abiotic firm surface, surrounded by a self-formed mucilaginous matrix.One of the prime examples is oral biofilm or dental plaque, which is a common phenomenon in the oral cavity of all mammals. The natural dentition and dental prostheses, including dentures and implants, are substrates for biofilms. The consumption of carbohydrate-rich food leads to increased secretion of organic acids by the bacteria forming oral biofilm or dental plaque. Subsequently, as the bacterial colonization increases, there is an overproduction of matrix polysaccharide alginate, and the mucoid biofilm facilitates resistance to antibiotics and to the host’s immune response. An extracellular polymeric substance (EPS) is produced by the bacteria, which causes chronic infection, and its treatment becomes difficult. Under untreated conditions, biofilm demineralizes the enamel, causing dental caries.2Biofilms were first formally defined in the mid-1980s; however, detailed knowledge on their mechanism of development was gained only recently.Formation of oral biofilm consists of five basic stages:5Entry of pathogenic microorganisms into the oral cavity.Congregation of pathogenic and normal microflora, and salivary pellicle formation.Marked increase of the biofilm-forming bacteria, formation of a mucilaginous layer and the beginning of dysbiosis, and the beginning of irreversible adhesion.Exchange of drug-resistant genes between pathogenic bacteria and normal oral microflora, along with completion of dysbiosis.Maturation of the biofilm and bacterial spread.The formation of a biofilm begins with the entry of pathogenic microorganisms into the oral cavity. This process is still reversible at this stage, after which these bacteria will slowly start forming a layer over the teeth and produce a mucilaginous slimy covering which is irreversible. This covering consists of extracellular polysaccharides, structural proteins, cell debris, and nucleic acids—referred to as EPS—which protects the bacteria in the biofilm from mechanical stress and antimicrobial drugs.5Biofilm acts as a predisposing factor for several oral infections, such as gingivitis, dental caries, periapical periodontitis, periodontitis, and peri-implantitis. Restorations, non-surgical or surgical periodontal therapies, root canal treatments, and dental implants are well established curative regimens that remove oral biofilm; however, these treatments are not always entirely successful at removing secondary biofilm infections. Biofilm management necessitates a very high rate of expenditure globally.5Topically applied antibacterial drugs become diluted and are not effective for an extended duration due to the complexity of the oral cavity and rapid clearance by saliva. A mature biofilm requires a superior concentration of antimicrobial agents for its removal in comparison to bacteria in planktonic biofilms. When antimicrobial agents are used, only some cells are exposed, while some of the drug entrance is reduced by the EPS matrix of the biofilm, hence diffusion of drugs into the deeper layer of oral biofilm is prohibited.3The oral cavity provides ideal conditions for microbial growth and proliferation by providing a moist, warm, and nutritious environment. Microbial colonization of pathogenic biofilm occurs due to the complex dynamic interactions among the host, microorganisms, and diet. The biofilm produces acid at the tooth restoration margin, leading to secondary caries, which is also the reason for most restoration failures. Pulp infections are also seen after dental restorations. Even after root canal therapy, persistent biofilms inside the root canal system result in constant apical periodontitis and re-infection. Dental implants and periodontal tissues are affected by the biofilm, leading to peri-implantitis and periodontitis. Therefore, oral rehabilitation procedures depend on the capacity of dental materials to incorporate specific antibiotic strategies for controlling and eliminating these infections, which is the long-term clinical accomplishment. A critical assessment of recently published, innovative antimicrobial strategies for managing oral biofilm-related infections are discussed in the following sections.4Bacteria surviving and growing within a biofilm exhibit a tremendous amount of antibiotic resistance. Their stable structural properties and the proximity of the bacterial cells within the biofilm create an excellent environment for horizontal gene transfer, which can lead to the spread of antibiotic-resistant genes amongst the biofilm inhabitants. As per the available literature, the following structural and physiological properties of biofilm-forming bacteria help them become tolerant to antibiotics and slowly become antibiotic-resistant.6The capsule is an important part of the biofilm; both Gram-positive and Gram-negative bacteria can have capsule thicknesses ranging from 0.2–1.0 μm. Biofilm adhesion and cohesion to a solid surface is by means of the capsule which uses Van der Waal, hydrogen bonds, and electrostatic forces, therefore contributing to the maturation of biofilm. The biofilm capsule consists of polysaccharides and glycoproteins that are influenced by conditions of different environments. The accumulation of antibacterial molecules by the capsule or glycocalyx layer constitutes up to 25% of its weight, resulting in the development of resistance to antibiotics and other antimicrobial drugs by the bacteria. The antimicrobial drugs are trapped in the adsorption sites of the glycocalyx matrix, where these drugs are degraded by the exoenzymes released by the MDR bacteria. Hence, the antimicrobial drugs slowly become ineffective in combating the bacteria present in the biofilm. 7Cell membranes are the major target site for major antibiotics; however, alteration or modification of cell membrane permeability and altered expression of porin proteins present in bacteria inside the biofilm do not allow the entry of antibiotics, thus leading to the development of resistance.7Efflux pumps present in the bacteria are responsible for antimicrobial resistance in biofilm structures. Generally, these efflux pumps are a group of transport proteins that help remove toxic and unwanted substances from the bacteria. These pumps do not allow antibiotics to enter the bacterial cell, leading to the emergence of MDR bacteria.8Plasmids are extrachromosomal genetic materials commonly found in biofilm-forming bacteria. Plasmids generally contain genes that are encoded for increased virulence by means of enzymes and proteins, leading to resistance to various antimicrobials and heavy metals. Sometimes these plasmids carry multiple resistant genes that are specifically resistant to most of the commonly used antibiotics, including macrolides, beta-lactams, aminoglycosides and fluoroquinolones. These genes usually get rearranged with the help of an inherent recombination system as seen with integrons and transposons. All these plasmids are conjugative; horizontal transfer of these resistant genes occurs in closely packed oral biofilm. In a slow but confirmative process, slowly all the bacteria become multidrug-resistant.9Regular exposure to antibiotics and other MDR bacteria results in the direct horizontal transfer of antibiotic-resistant genes and leads to long-term genetic adaptations or mutations in response to an alteration in their growth environment. Such mutations lead to the development of drug-resistant strains.9Several bacteria regulate their metabolic/physiological activities through a mechanism called quorum sensing, where the individual bacteria communicate with each other by releasing diffusible components. Quorum sensing facilitates interactions among bacteria, which leads to host colonization, the formation of biofilms, and their defense against antimicrobials, the host defense system, and their adaptation to an ever-changing environment. Clearly, a degree of quorum sensing in pathogenic bacteria is responsible for their virulence and breaking down the host defenses.10Differences in oxygen availability and nutrients inside the biofilm affect the metabolic action and growth rate of the bacteria. Varying concentrations of metabolic substrate and by-products affect the growth of bacteria within the biofilm, which leads to heterogeneity in the growth of the microbial population. The available nutrients and fluctuating concentration of oxygen affect the metabolic activities of cells in the peripheral region of biofilm that supports the proliferation of different species of bacteria. The metabolically active bacteria are killed by biocides, whereas the dormant bacteria which are less vulnerable to the antimicrobial drugs survive for a long period.10The antimicrobial agent-tolerant cells, called persisters, are responsible for severe chronic infections. The major challenge in most dental clinics is the detection of bacterial strains. The lowering of the ATP level decreases antibiotic target activity, leading to the formation of persisters. The resistant persister cells in the bacterial biofilm exhibit antibiotics tolerance.11Salmonella enteritis and Escherichia coli. The stress response also helps in their cellular damage repair mechanism. Stress may be induced for several reasons, including deprivation of nutrients caused by the stationary phase in the growth of bacteria, at high or low temperatures, acidic pH, and higher osmolality.11The stress response in a biofilm is attributed to the change in the structural and physiological functions of bacteria that lead to an increase in their stress tolerance capacity. Normally, the formation of the cell envelope and synthesis of thin aggregative fimbriae are controlled by a stress response in A number of different articles in the scientific literature describe the following techniques for effective management and removal of oral biofilms.19The use of antimicrobial materials is vital to prevent bacterial adhesion and formation of a biofilm, as most infections in the oral cavity originate from it. Extensive efforts have been made to provide dental materials with antimicrobial properties, aimed antimicrobial agent release, contact killing, and multifunction.12Antiplaque agents such as essential oils  and surfactants (sodium lauryl sulphate) have been effective in removing dental plaque. Antimicrobial agents such as bisbiguanides, metal ions, phenols, and quaternary ammonium compounds have been successfully formulated along with the addition of antiplaque agents into toothpaste and mouthwash for control and removal of oral biofilms/dental plaque.14A very specific technique is used to kill the bacteria inside the biofilm; antimicrobial agents are preloaded and incorporated in the dental materials used for various dental treatments. Antibiotics, in a combination of silver compounds, were the first kind of antimicrobial agents used for biofilm removal. The main advantage of this technique is that even at very low concentrations, antimicrobial agents against biofilms are released to prevent bacterial growth. In addition, using a antimicrobial release agent provides a high local dose at the site of interest, strong broad-spectrum antimicrobial activity, reduced systemic toxicity, and minimal risk of antimicrobial resistance. However, this technique also has some disadvantages: it is short-acting, and exhaustion of the antimicrobial reservoir in the dental material can occur. Currently, nanotechnology is being used to incorporate various antimicrobial agents into dental materials, providing a better antimicrobial effect, and for a longer duration.12The contact-killing strategy is based on the strategic incorporation of a wide range of antimicrobial agents  into dental materials. The compounds are non-toxic and can be easily mixed with dental materials to form a covalent compound structure. These are broad-spectrum antimicrobial agents that have a strong effect on both Gram-positive and Gram-negative bacteria when they both come in contact with the dental material. This strategy has several advantages, being non-toxic, with long-term anti-microbial activity and non-irritant properties, while drawbacks include the fact that it has a bacteriostatic instead of a bactericidal effect, and that surface biofouling occurs (roughness on the surface of the teeth).12Antimicrobials with multiple or broad-spectrum activities have been preferred over the years for increasing the efficacy of controlling oral infections caused by various bacteria, viruses, and fungi. Silver compounds with free silver, silver nanoparticles (AgNPs), have a high surface-to-mass ratio, and the control of releasing kinetics is easier, as is the long-term maintenance of the antibacterial effect. In experimental dental adhesives and composites, very low concentrations of AgNPs are effective against plaque biofilms. The combined action of AgNPs and quaternary ammonium salts to enhance the antibacterial effects has been recently adopted for the preparation of antimicrobial dental materials. Antimicrobial dental material provides effective killing of bacteria on its own surface, as well as away from it. While such materials are multifunctional, there are currently not enough combinations available that provide synergistic and enhanced antimicrobial properties, and more research is needed.12Removal of oral biofilm using nanoparticles has enormous potential. Nanoparticles have excellent antibacterial activity and can be used to target specific biofilm-forming microorganisms without disturbing the normal microflora of the oral cavity. However, their use is expensive and cannot be regularly practiced in dental clinics. Administering nanoparticles requires great precision and may lead to severe side effects. Furthermore, the biomimetic properties of nanoparticles should be highly precise and target-specific to achieve the desired results.15Photodynamic therapy (PDT) is an emerging non-invasive technique that is employed against oral infections, especially periodontal infections. Photodynamic therapy involves an oxygen-dependent photochemical reaction that occurs upon light-mediated activation of a photosensitizing compound leading to the generation of cytotoxic reactive oxygen species, predominantly singlet oxygen. The therapy is directly applied topically to the periodontal pockets, together with an antimicrobial agent for the removal or killing of the microorganisms. Factors such as overdose and exposure duration should be always taken into consideration to minimize side effects and retain the normal microflora. Photodynamic therapy also minimizes the emergence of MDR strains.16in vivo applications without destroying the surrounding tissues and microflora. Apart from the removal of dental plaque, CAP has been used in bleaching, endodontic treatment, sterilization of surgical instruments, and composite restoration. However, more studies are needed to assess the ease of CAP usage and to increase its field of application.17Cold atmospheric plasma (CAP) is a non-invasive technique used in dentistry and oncology for its antimicrobial and cell necrosis properties. Non-thermal plasma at a temperature of less than 104°F is administered at the application site. This method uses a highly reactive mix of ions and electrons, radical species, molecules in the ground or excited state, and quanta of electromagnetic radiation (UV photons and visible light). Treatment with CAP is carried out under normal atmospheric conditions, which makes it easier for Salvadora persica, Allium sativum, Punica granatum, as well as naturally occurring compound honey have been reported for their biofilm removal properties. One of the most important aspects of herbal products or naturally occurring compounds is their complex chemical structure, for which bacteria fail to develop resistance. However, their therapeutic action can be slow, and sometimes the ideal dosage versus toxicity may become a concern. Nevertheless, they can serve as an excellent adjunct in combination with antibiotics, or other antimicrobials, with a good synergistic effect. Hence, more natural and herbal compounds should be explored for the treatment of oral biofilms.19Plants such as There are consequences to the global widespread use of antibiotics at variable concentrations. Such use may not only affect the microbiota; variable genetic effects can also occur, such as antimicrobial resistance, a mutation that has a direct effect on humankind. Furthermore, biofilms are already highly defensive and resistant to the action of antibiotics.Several therapeutic approaches have been utilized over the years for the effective management of oral biofilms and related infections. However, the development of MDR bacteria has made many of these procedures ineffective. Moreover, some of these procedures are also detrimental to the normal flora present in the oral cavity. In light of this, it is very important to use the correct combination of treatment procedures and materials, which are not only more target-oriented, but also have fewer side effects.


The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and  In the past 20 years, there have been significant advances in the research and understanding of the biology of the placenta and its derivatives. Initially, the placenta drew attention as an interesting cell source due to its early embryological origin suggesting that cells derived from the placenta could possess unique plasticity and differentiation properties . In addiWe now know that perinatal derivatives are promising for a wide range of regenerative medicine applications due to their differentiation capabilities but mainly due to their unique immune modulatory properties. As a matter of fact, many preclinical studies and initial clinical trials have demonstrated that perinatal derivatives may represent important tools for restoring tissue damage or promoting regeneration and repair of the tissue microenvironment . The terFirst International Workshop on Placenta-Derived Stem Cells was published  . Even toin vitro culture. For example, we previously demonstrated that genomic Polymerase Chain Reaction amplification was not able to determine the presence of maternal cells in the freshly isolated mesenchymal fractions of both the amnion and chorion  and human amniotic membrane epithelial cells (hAEC) from placenta, it has previously been demonstrated that cell culture up to passage 4 after isolation (passage 0) can induce changes in the expression of cell markers. Such changes include the significant increase in adhesion molecules  and the significant decrease of CD14, CD45, and HLA-DR expression on hAMSC, as well as the significant increase of CD13, CD44, CD105, CD146 expression on cultures of hAEC .in vitro characterization and clinical application of PnD.Here, considering the numerous publications and the increasing interest in perinatal derivatives, we address specific issues that are relevant for the clear and precise definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. In addition, we describe where the cells are located within the placenta and provide an atlas of the human placenta. We also describe cell morphology and phenotype and propose nomenclature for the cell populations and derivatives discussed herein. The proposed nomenclature will be crucial to lay the foundation for the consistency in the scientific community when referring to PnD. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing, The placenta is the first organ to develop in mammals. It is essential for the successful growth of the embryo, and later the fetus, and its crosstalk with the uterine maternal compartment is indispensable. Its fundamental role is underlined by the fact that impaired formation of placental tissues leads to pregnancy disorders, such as preeclampsia, fetal growth restriction, recurrent miscarriage, and stillbirth. The embryo-maternal interface is based on an intimate and controlled relationship between the conceptus and the mother. The placenta and extraembryonic membranes maintain this essential contact for supporting the development of the new organism by acquiring oxygen and nutrients, eliminating waste, and avoiding immune rejection. However, despite its importance in reproductive outcome, there is still a limited understanding about human placental development, mainly due to ethical and logistic difficulties in investigating it in the early stages, as well as in extrapolating data from other species .The development of the human placenta begins from the first days after conception . Fertilith day post conception, some cells from the epiblast migrate and position between the cytotrophoblast and the underlying embryonic disk, creating a small space that will later become the amniotic cavity. The cells derived from the epiblast, which surround the future amniotic cavity, are called amnioblasts and will give rise to the amniotic epithelium. Subsequently, the cytotrophoblast secretes a spongy layer of acellular material, called the extraembryonic reticulum, which will give rise to extraembryonic mesoderm after invasion of the migratory cells from the epiblast. This layer surrounds the yolk sac and the amniotic cavity and, subsequently, will constitute the amniotic and chorionic mesoderm (extraembryonic somatopleuric mesoderm). At this point fluid-filled spaces (lacunae) begin to develop within the syncytial mass that enlarge and merge leading to the formation of a lacunar system (lacunar stage) (As shown in r stage) . As a cor stage)  the cytor stage) . From thr stage) , the mesDuring the first trimester of pregnancy, the placenta develops all the structures needed for a sufficient supply of nutrients to the embryo and, subsequently, the fetus during pregnancy . SeveralFrom the chorionic plate, larger villi grow into the intervillous space  and formThe placental villi are covered by the syncytiotrophoblast that comes in direct contact with maternal plasma/blood. Directly underneath the syncytiotrophoblast, a complete layer of villous cytotrophoblast can be found . These cDuring the first half of the first trimester of pregnancy, the freely floating villi are very primitive and are classified as mesenchymal villi. At about 8 to 10 weeks of pregnancy, the first mesenchymal villi differentiate into immature intermediate villi, characterized by stromal channels where placental macrophages can easily be visualized. The placental villi that connect to the uterine wall are called anchoring villi . This isAt the end of pregnancy, the placenta has all the structures that were needed to supply the fetus with sufficient amounts of nutrients and gases, as well as to allow excretion of waste products . In the The fetus bathes in the amniotic fluid. The outer border of the fluid-filled cavity is demarcated by the amniotic epithelium, a cuboidal and mostly single layered epithelium . Under tThe next layer is the chorionic mesenchyme. This connective tissue is vascularized and contains the vessels between placental villi and umbilical cord. At the site of the decidua basalis, this layer is called chorionic plate , while aAt term, the anchoring villi are still attached to the uterine wall . HoweverAt the end of pregnancy, trophoblast that has invaded into uterine spiral arteries  and utero-placental veins (endovenous trophoblast) can still be found. Invaded endometrial glands are hardly visible at term. In addition, at term the endoarterial trophoblast has led to a widening of uterine spiral arteries to allow a constant flow of maternal blood into the intervillous space . MaternaSeveral cell types can be obtained and expanded from the different regions of the human placenta and the fetal annexes. Human placenta cells (hPC) is a generic term used to refer to any type of cell that can be isolated from term placenta; the most prominent being epithelial cells, mesenchymal stromal cells (MSC), endothelial, and hematopoietic cells.First International Workshop on Placenta-Derived Stem Cells held in Brescia, Italy in 2007. This includes the expression of CD90, CD73, and CD105, and the lack of expression of CD45, CD34, CD14, and HLA-DR  is a general term commonly used to refer to MSC from various perinatal tissues. hPMSC from the different tissues described herein possess similar characteristics in accordance with the minimal consensus criteria reported for MSC from other adult tissues, such as bone marrow  and estad HLA-DR .In order to advise the scientific community on the precise localization and nomenclature of perinatal tissues and cells, in the following paragraphs the main characteristics of the different placental/perinatal regions/tissues, and features of their cells will be described. To discriminate similar cells of different origins more accurately a nomenclature will be proposed . We alsoThe human amniotic membrane (hAM) represents the wall of an embryo/fetal annex called the amnion or amniotic sac, which encloses the amniotic cavity and contains the amniotic fluid (AF), . During in situ investigation revealed the morphological heterogeneity of the different cell populations belonging to different regions identified within the hAM  with microvilli on the apical surface in direct contact with the AF . An in s the hAM . This he the hAM . InteresHuman amniotic membrane cells (hAMC) are of fetal origin. The structure of the hAM defines a layer of hAEC in direct contact with the AF and a population of amniotic membrane mesenchymal stromal cells (hAMSC) embedded in the underlying connective tissue . In addiAmniotic cells can be isolated from the hAM either as a heterogenous total population or by separate enzymatic digest to obtain hAEC and hAMSC. In most protocols trypsin is used for isolation of hAEC and collagenase for hAMSC . HoweverThe quality of amniotic cells mostly depends on expansion conditions rather than isolation methods. They can be expanded using standard culture media composed of a basal medium, supplemented with serum, antibiotics and antimycotics. Some groups use additional supplements including epidermal growth factor (EGF)  or speciThe hAM epithelium is the cellular layer directed towards the fetus and in touch with the AF . hAEC loGenerally, isolated primary hAEC are heterogeneous for their expression of surface antigens and also show varying differentiation capabilities , indicathAEC express markers that are otherwise found exclusively in undifferentiated embryonic stem cells, embryonic carcinoma, and embryonic germ cells , Miki e. In conthAEC in different regions of the hAM may differ in their functional status, such as metabolic and secretory activity ; howeverHuman amniotic membrane mesenchymal stromal cells are embedded in the rich extracellular matrix of the hAM  and afteThe chorionic membrane (hCM) represents the fetal component of the placenta. Cells from the hCM are generally referred to as human chorionic membrane cells (hCMC). It is formed by extraembryonic mesoderm and the two layers of trophoblast (syncytiotrophoblast and cytotrophoblast) that surround the embryo and other membranes. The chorionic villi emerge from the entire chorion during the first phases of embryonic development leading to the formation of the primitive placenta, whereas later on they reach the highest level of development and branching in correspondence of the chorion frondosum which rises from the chorionic plate (hCP) and which forms the definitive placenta together with the basal part of the maternal decidua. The rest of the chorion, where villi have turned atrophic, becomes smooth and subtle, does not take part at the formation of the placenta and is called chorion laeve (hCL).Very early in pregnancy, the chorion of the human placenta starts as a double layer of trophoblast with the syncytiotrophoblast covering the early intervillous space and a layer of cytotrophoblast (hCTB) in second row, proliferating, differentiating and maintaining the syncytiotrophoblast (hSTB) via syncytial fusion. At the end of the second week after fertilization (day 12 p.c.) a new layer of extraembryonic mesenchymal cells (hCMSC) develops and covers the cytotrophoblast towards the embryonic side. Thus, the mesenchymal cells are added as a new border to the original blastocyst cavity without a specific epithelial coverage. At that time of gestation, the amniotic sac is still very small and not in contact with the chorion.Since first development of placental villi takes place all over the surface of the chorion, the chorion frondosum, i.e., the part of the chorion where placental villi are found, is not restricted to a specific part of the chorion. At this time of placental development, the entire chorion is chorion frondosum and the placenta proper develops as a ball-shaped organ. Only at the end of the first trimester, with the onset of maternal blood flow into the placenta, villous tissues at the abembryonic part of the chorion regress, leading to a smooth chorion, the chorion laeve. The remaining part of the chorion frondosum develops into the chorionic plate covering the placenta proper towards the fetus until delivery. Hence, with the beginning of the second trimester, the placenta changes its shape towards a disk-shaped organ.In literature, there are descriptions of the general features and characteristics of mesenchymal cells from term chorion, not differentiating between chorionic plate (hCP-MSC) and chorion laeve (hCL-MSC). Therefore, this paragraph displays the general features of chorionic mesenchymal stromal cells (hCMSC), not differentiating between the two sites.Several publications have dealt with various isolation methods of placental mesenchymal stromal cells and the comparison of these cells isolated from different placental sites, with the intention to find the most suitable ones. Isolation techniques vary from explant methods , enzymatDuring early development of the embryo, the very first cell lineage of the human is formed. At around day four after fertilization, the blastocyst develops from the morula by differentiation of a cell layer, the human trophectoderm (hTED). This is the forerunner of all trophoblast cells and layers throughout pregnancy. At the time of implantation, around day 6-7 after fertilization, trophectoderm cells in contact with the embryoblast attach to the uterine epithelium. Cells in direct contact with the epithelium fuse and generate the first trophoblast cell type, the syncytiotrophoblast (hSTB) that penetrates through the uterine epithelium. This first multinuclear primitive syncytium is the first invasive placental component that expands into the maternal compartment. The other trophoblast cells remain as undifferentiated human mononuclear cytotrophoblast (hCTB) cells, characterized by an epithelium-like phenotype with cuboidal cell shape .Three weeks after fertilization placental villi are fully developed. The hSTB has differentiated into the outermost cover of placental villi, now serving as the epithelial layer in direct contact with maternal blood. Below the hSTB, proliferative hCTB make up the second epithelial layer of the placental villi. The villous cytotrophoblast (hVCTB) maintains the hSTB by continuous proliferation, differentiation and fusion with the hSTB . With thThe syncytiotrophoblast is a multinucleated and polar layer without lateral cell borders, not showing any proliferative activity. The syncytiotrophoblast only has an apical membrane facing maternal blood and a basal membrane in contact to the underlying villous cytotrophoblasts . The maiTrophoblastic cell columns at the tips of anchoring villi attached to the maternal decidua give rise to extravillous trophoblast (hEVT) cells. Proliferative proximal cell column trophoblast (hpCCT) sitting on its basal lamina represents the progenitor cell pool of differentiated hEVT. In the distal cell column, the cells differentiate into non-proliferative distal cell column trophoblast (hdCCT). Some of these cells undergo polyploidization and senescence upon differentiation into hEVT . In geneThe cell column trophoblast undergoes a multi-step differentiation process with epidermal growth factor receptor-positive (EGFR+) hpCCT, characterized by high proliferative activity. These cells further differentiate into non-proliferating human leukocyte antigen G-positive (HLA-G+) hdCCT, ready to invade the decidua. HLA-G is expressed in all extravillous trophoblast . EGFR ishEVT can be found at various sites of the feto-maternal interface, such as the chorion laeve, the chorionic plate, cell islands within the villous tissues, and in the uterus at the site of the placental bed. Most of these cells invade into the maternal uterine stroma and the inner third of the myometrium as interstitial extravillous trophoblast (hiEVT) . From thin vitro studies succeeded in deriving hTSC from blastocysts and primary hCTB preparations , which is abundantly expressed in early first trimester, but becomes rapidly downregulated and restricted to individual hVCTB towards the end of the first trimester (Trophoblast stem cells (hTSC) are the progenitors of the differentiated hCTB in the placenta. They have been isolated from both the chorionic membrane and villous tissue of the placenta [reviewed by arations , as wellarations . A reasoarations , promotirimester . TEA Domrimester . Molbay rimester . Interesrimester . Solublerimester . TherefoInterestingly, a recent study used trophoblast organoids to generate hTSC . They deThe chorionic plate (hCP) of the human placenta is built up of layers of two different origins, chorion and amnion. The amnion is located towards the fetus and towards the intervillous space the chorion frondosum is found, from which placental villi grow into the trophoblast lacunae. Also, within the chorionic plate the amniotic membrane is an avascular tissue.The chorion frondosum differs from the chorion laeve in a variety of aspects. It is a spongy layer with few mesenchymal cells and clefts in direct contact with the amniotic membrane. This layer is followed by a thicker layer of compact mesenchyme, in which the chorionic plate blood vessels from the fetus to the placenta (and vice versa) are located. This layer ends with a rudimentary basement membrane towards the intervillous space. At this site, remnants of the complete layering of the chorion early in pregnancy can be found, including small parts of the syncytiotrophoblast and nests or single extravillous trophoblast cells. Since the cytotrophoblast cells are located outside the villous part of the placenta, they are termed extravillous trophoblast. Also, throughout pregnancy more and more fibrinoid covers the surface of the chorionic plate in direct contact with maternal blood.The cells that are located in the chorionic plate belong to the amnio-chorionic membrane as well as to the chorion frondosum . The celin vitro cell culture passaging of MSC isolated from the chorionic plate and chorionic villi led to a high risk of overgrowth by maternal MSC of the placental decidua  display a round to polygonal phenotype with hyperchromatic nuclei showing irregular shapes. Although in general the hCP-EVT display the same phenotypic characteristics as the hEVT in the decidua, they are generally smaller and show less variation in their size and shape. They are HLA-G positive and do not display any signs of proliferation, migration or invasion.In the human placenta, the chorionic villi are arranged as villous trees that are connected via a major trunk to the chorionic plate. From the major trunk, a stem villus, multiple branches develop into intermediate villi, finally ending in free-floating terminal villi .All chorionic villi of the human placenta share the same general morphological structure. They have a core of mesenchymal cells derived from extra-embryonic mesoderm. The core structure of all villi contains vessels from large arteries and veins in stem villi down to arterioles and venules in intermediate villi and capillaries and sinusoids in terminal villi. The extracellular matrix is composed of a large number of reticular and collagen fibers.The vessels are surrounded by MSC that show different levels of differentiation from terminal to stem villi. Also, macrophages (positive for markers such as CD68 and CD163) can be found in the villous stroma of chorionic villi. These macrophages, referred to as Hofbauer cells, are derived from two different sources, directly developing from placental mesenchymal cells in the villous stroma or deriving from circulating fetal monocytes.The outer cover of the chorionic villi is organized as a two-layered epithelium. The inner layer is composed of mononucleated villous cytotrophoblast (hVCTB) cells that rest on a basement membrane separating the trophoblast cells from the villous stroma. The hVCTB cells proliferate and their daughter cells differentiate to finally fuse with the overlying cover, the hSTB, that is a true syncytium that comes into direct contact with maternal blood circulating around the chorionic villi.Within chorionic villi, fetal cells build the epithelial layer as well as the villous core of the villi. The surrounding epithelium is built by a continuous layer of syncytiotrophoblast, in direct contact with the maternal blood, and an underlying layer of mononucleated villous cytotrophoblast. Separated by the trophoblastic basement membrane the villous stroma is filled with mesenchymal cells, macrophages, and vessels formed by endothelial cells, pericytes and smooth muscle cells.MSC of the chorionic villi express MSC markers  in accorThe villous trophoblast as a tissue can be divided into two types of cells/layers: mononucleated cytotrophoblasts and the multinucleated syncytiotrophoblast. It needs to be stressed here that in a given placenta, there is only one syncytiotrophoblast covering all chorionic villi of that placenta .The layer of the mononucleated villous cytotrophoblast is the basal and germinative layer of the villous trophoblast compartment. The cells rest on the basement membrane underneath the villous trophoblast layers. Villous cytotrophoblast cells change their morphology during pregnancy. During the first trimester of pregnancy, the cells display a cuboidal shape and form a nearly complete layer. At term, the cells display a flattened phenotype, separated from each other but connected to each other by long cytoplasmic extensions. A subset of the cells proliferates throughout pregnancy, which may point to a small subset of progenitor cells within this layer. Also, some of the cells may display their progenitor status as it has been shown that they can be induced to differentiate towards the extravillous lineage .The syncytiotrophoblast is a continuous, multinucleated layer without lateral cell borders. Hence, a single syncytiotrophoblast covers all villi of a single placenta. The apical membrane of the highly polarized syncytiotrophoblast shows microvilli to amplify seven-fold the surface  for a beThe chorion laeve of the human placenta hCL,  developsThe chorionic part of the chorion laeve is organized into a thin compact stromal layer, densely packed with collagen fibers and containing only few scattered mesenchymal cells, and a fibroblast layer with mesenchymal stromal cells hCL-MSC,  includinTowards the uterus in touch with the chorion laeve is the only maternal layer of this part of the placenta, the capsular decidua (hCD). This layer includes decidual cells as well as some smaller vessels plus mobile cells such as macrophages and lymphocytes. It needs to be stressed that at the time of delivery the capsular decidua or decidua capsularis is firmly attached to the parietal decidua or decidua parietalis and, hence, some parts of the decidua parietalis may be associated with the decidual layer of the chorion laeve after delivery.in vitro into cardiomyocytes and express genes associated with heart morphogenesis and blood circulation including serotonin receptor B2 (HTR2B)  . hCL-MSC (HTR2B) .Human chorion laeve mesenchymal stromal cells have also been shown to secrete cytokines quite common to MSC, such as Insulin Growth Factor-1 (IGF-1), VEGF, Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), Angiopoietin 1 (Ang-1), and TGF, with partly contradictory data about lower or higher secreted levels compared to hMSC derived from amnion, umbilical cord, chorionic villi or decidua parietalis .The extravillous trophoblast of the chorion laeve (hCL-EVT) displays a round to polygonal phenotype and irregularly formed nuclei that are hyperchromatic. Also, the hCL-EVT cells are generally smaller than the hEVT in the placental bed. Interestingly, although hCL-EVT are HLA-G positive, they do not show signs of invasiveness, different to their counterparts in the placental bed. It has been speculated that local factors keep these cells in a non-invasive state. Isolation of hCL-EVT has been performed and published .funiculus umbilicalis) is a conduit that connects the placenta to the developing embryo/fetus and is responsible for exchange of nutrients and gasses during gestation. The human umbilical cord is formed when the body stalk  and the vitelline duct  deriving from the yolk sac plus the umbilical coelom are enveloped by the spreading amnion between the fourth and the eighth week. During the third month of gestation, numerous elements degenerate: the vitelline duct (it can remain in the form of a Meckel’s diverticulum); the allantois ; the vitelline circulation system in the extra-embryonic region; the umbilical coelom, which clumps and disappears. In humans, the umbilical cord is approximately 50 cm long and 2 cm in diameter at term and is normally attached in the middle of the placenta.In placental mammals, the umbilical cord (UC) , referred to cell populations present within the wall or in the perivascular region of umbilical vein and arteries contained in the umbilical cord  share common features with fetal epidermal keratinocytes in terms of the expression patterns of cytokeratins, cell surface markers, and their differentiation potential . UmbilicPhenotype characterization of hUC-AEC has shown expression of CK8, CD14, and CD19, markers associated to simple epithelium and skin stem cells. Immunophenotype analysis has shown that hUC-AEC are negative for CD45, CD90, CD105, whereas they are positive for CD29, CD44, CD49f, CD166. One study reported that a small fraction of hUC-AEC display stem cell-specific molecules such as SSEA-4 and TRA-1-60 .Considering the embryological development of the umbilical cord, the stromal component of the amniotic membrane is completely fused with the connective part of the umbilical cord. For this reason, it is difficult to distinguish and isolate a cell population of amniotic origin with mesenchymal characteristics  also follow the minimal phenotype and differentiation criteria of the consensus paper by Parolini et al., , and expAs mentioned above, the cord lining membrane mesenchymal cells are fibroblast-like cells with spindle-shaped morphology  that meeInterestingly, hUC-saWJ-MSC can also display epithelial cell properties due to the expression of cytokeratins CK1, CK7 and CK14, the epithelial cell marker MUCIN1 (CD227), and the epithelial cell-to-cell adhesion molecule CD151 .For details on this cell population please refer to the cell population described in the paragraph on hUC-WJ-MSC .Almost 45% of the cells resident in the Wharton’s jelly reside in the perivascular region . UmbilicIn vitro culture of human umbilical cord vascular smooth muscle cells (hUC-VSMC) promotes a switch from a contractile (quiescent) phenotype to a more secretive (proliferating) one  one . In manying) one  and are ing) one .The differentiated myofibroblasts represent the functional phenotype of Wharton’s jelly. Mesenchymal cells that comprise the functional myofibroblasts of the stroma and their precursors are found in this unusual connective tissue. These have a fibroblast-like morphology and can be identified in five stages of differentiation based on their sequential and additive expression of vimentin, desmin, αSMA, γ-SMA, and smooth muscle myosin .As mentioned earlier, the umbilical cord comprises two fetal arteries and one fetal vein. The umbilical arteries transport low-oxygenated blood, loaded with metabolic waste products, from the fetus via fetal arteries of the chorionic plate into the placental villous tree, which mainly consists of (1) stem villi with centrally located smaller arteries and veins, and a paravascular capillary network; (2) intermediate villi with arterioles, venules, capillaries; and (3) terminal villi with sinusoids and capillaries. The intervillous space is filled with maternal blood, supplied by afferent maternal spiral arteries penetrating the basal plate and efferent maternal utero-placental veins. The feto-maternal exchange takes place across the vasculo-syncytial membrane of the terminal villi. There, the fetal blood is loaded with oxygen and nutrients and transported via the venous system of intermediate and stem villi, veins of the chorionic plate and the umbilical vein back to the fetus .in situ studies evidenced heterogeneity of the endothelium of the umbilical cord and placental blood vessels , respectively). HUAEC express higher levels of plasminogen activator inhibitor-1 (PAI-1), Cx40, 17β-Hydroxysteroid dehydrogenases (17β-HSD2), and vascular cell adhesion molecule 1 (VCAM-1); and lower levels of vWF and estrogen receptors β when compared to HUVEC. HUVEC and HUAEC differ in their expression of angiotensin converting enzyme, endothelin-1 and endothelial nitric oxide synthase (eNOS) activity  are polygonal cells with a smooth surface and grow in loose arrangements and forming monolayers with cobblestone morphology. They express artery-related genes  and more endothelial-associated genes than human placental venous endothelial cells (hPV-EC). VEGFs induce a higher proliferative response on hPA-EC, whereas placental growth factors (PlGFs) are only effective on hPV-EC. In vitro, hPV-EC are spindle-shaped cells with numerous microvilli at their surface. They grow closely apposed to each other, form fibroblastoid swirling patterns at confluence and have shorter generation and population doubling times than hPA-EC. hPV-EC over-express development-associated genes , and show an enhanced adipogenic and osteogenic differentiation potential unlike hPA-EC  gradually increases and separates the cells of the epiblast (embryo) from the amnioblasts surrounding the embryo in the newly formed amniotic cavity. The hAF allows for fetal movements and growth inside the uterus. It also allows for the exchange of different nutrients and chemicals between the fetus and the mother .in vitro and in vivo stimuli suggests their stem cell origin (Human AF cells (hAFC) can be isolated during the three trimesters of gestation , and havl origin . In 2004l origin .in vitro long-term culture. In addition, MSC from the AF (hAF-MSC) have also been described.Human amniotic fluid cells are a heterogeneous population that can be classified into three groups according to their molecular, morphological, and growth characteristics : (1) epiPrevious studies on hAFC detected cell markers from all three germ layers  proved tOn one hand, hAFSC share many characteristics with MSC such as the positivity for CD73, CD90, CD105, and MHC class I, and lack of MHC class II, CD40, CD80, and CD86 , with loin vivo or grown in adhesion, and their natural environment changes from a suspension fluid to a flat surface. Although the antigen surface expression is detected at the first passages  and, strikingly, human cells contributed to the formation of glomerular structures, differentiating into podocytes  are plastic adherent cells defined following the minimal criteria of the consensus paper by The decidua appears in mammals (including humans) with hemochorial placentation and an invasive trophoblast. The human decidua (hD) derives from the endometrium and is therefore of maternal origin. After ovulation, during each menstrual cycle, a reaction called decidualization develops. This process of differentiation involves structural and functional changes in all cells of the endometrium. If menstruation occurs, this tissue is discarded; however, if pregnancy takes place, decidualization continues through the effect of pregnancy hormones . Three tHuman basal decidua or decidua basalis (hBD) is located between the myometrium and the chorionic plate. The basal decidua is a thin plate of maternal endometrial tissue to which the anchoring villi are attached and which is invaded by extravillous trophoblast. The basal decidua modulates the allocation of maternal-fetal resources. The vessels in the basal decidua supply maternal arterial blood to the intervillous space between the fetal chorionic villi and receive venous blood from the placenta . ImportaHuman capsular decidua or decidua capsularis (hCD) is the thin layer of decidua that encapsulates the surface of the chorion laeve towards the uterine lumen. With the growth of the fetus, the capsular decidua is stretched and eventually fuses with the parietal decidua, thereby obliterating the uterine cavity .Human parietal decidua or decidua parietalis (hPD) is the deciduous layer that covers the rest of the uterine cavity. It contains maternal blood vessels and lymphatic vessels to maintain the nutritional and metabolic balance in the maternal compartment. From the third month of pregnancy, the hCD and hPD merge together as a consequence of membrane and fluid increment, as well as of fetal growth and development.Histologically, the hD is composed of decidual stromal cells DSC, , glandulDecidual stromal cells (DSC), the main cellular component of the hD, exert activities that are thought to play a key role in embryo implantation, trophoblast expansion, and the development of fetal-maternal immune tolerance. These cells originate from fibroblastic precursors located around the vessels and are detected in both the endometrium and decidua. During the luteal phase of the menstrual cycle, under the effect of the ovarian hormones estradiol and progesterone (P4), a predecidual reaction begins around the vessels and spreads through two thirds of the endometrium facing the uterine cavity. Precursors of DSC (preDSC) leave the vessels and differentiate into decidualized cells, which exhibit a rounder shape and secrete prolactin (PRL) and other factors such as insulin-like growth factor-binding protein 1 (IGFBP1) and IL-15, and express dickkopf WNT signaling pathway inhibitor 1 (DKK1), and forkhead box protein O1 (FOXO1). When menstruation occurs, these differentiated cells are discarded; however, if pregnancy takes place, this process of differentiation  continues through the effect of pregnancy hormones .There is a great deal of confusion in the terminology related to DSC, but what must be considered is that in both the decidua and endometrium, DSC can be found as undifferentiated and differentiated  cells; undifferentiated human DSC are referred to as hD-preDSC , and decDecidual stromal cells can be obtained from first-trimester decidua (elective termination of pregnancy)  or thirdin vitro in the presence of P4, as observed in the case of basal (hBD-dDSC) and parietal (hPD-dDSC) human decidualized decidual stromal cells by the secretion or expression of PRL and IGFBP1  show a phenotype similar to that of the first trimester hD-preDSC . Likewisd IGFBP1 . In singd IGFBP1 .in vitro culture in DMEM  of decidual tissue followed by  in DMEM  or Opti- in DMEM . The mai in DMEM  and othe in DMEM . The resMesenchymal stromal cells that meet the minimal consensus criteria  can be iMost studies on hDMSC have been based on cells isolated from the basal decidua (hBD-MSC) , althougBy retroviral transfer of reprogramming factor genes, hBD-MSC have been reprogrammed into pluripotent stem cells that maintain a normal karyotype, share similar characteristics to human embryonic stem cells, and differentiate into the three mesenchymal lineages . hPD-MSCIn addition, the expression of pericyte-associated antigens such as 3G5, STRO-1, CD146 and αSMA suggested that hPD-MSC may be derived from vascular stem cells . Interesquasi impossible task due to the heterogeneity of the starting material given by varying isolation and culture protocols. In addition, even though there are a few reports claiming that perinatal cells express markers that are not present in all perinatal regions, a detailed comparison and confirmation by other research groups is needed.Herein we addressed a few major challenges in the field of perinatal derivatives. First, we provide a thorough description and mapping of the human placenta, including a description of the different cell populations and clarification of where they are located within perinatal tissues. Second, given that the names and abbreviations used to refer to the different cell populations are often misleading and even inappropriate, we propose nomenclature for perinatal tissue and cells. Another important challenge relates to cell isolation and culture/expansion because specific characteristics, such as phenotype, change during culture and can depend, in many cases, on culture conditions. The literature analysis we performed did not allow us to identify specific markers to uniquely identify the cell populations discussed herein. At the current time, this is a A widespread comparison between isolation protocols, culture conditions, and cell phenotype, remains a critical challenge to be addressed. Therefore, we propose that consortiums active in the field work together to address this challenge. We also propose the adoption of the herein proposed nomenclature based on the precise tissue and cell localization. A defined starting material is the first step towards better data interpretation, study comparison and ultimately, for the establishment of standard cell preparation protocols for clinical use .That having been said, a concerted effort is still required to standardize perinatal cells. Even though the current cell heterogeneity doesn’t seem to be a limiting factor for functionality and therapeutic efficacy, since preclinical studies and initial clinical trials have demonstrated efficacy of different perinatal cells. Rather, standardizing cells and deciphering cell heterogeneity is of utmost importance to potentially fine-tune cells for specific therapeutic applications and to select cells that will provide an optimal response to the disease.AS, OP wrote the Introduction. FA, MB, RDP wrote section “Development of the Early Placenta”. RDP, BH wrote section “Structure of the Early Placenta”. RDP, FA wrote section “Structure of the Term Placenta”. All authors contributed to section “Cells Isolated from Term Placenta”. ILO wrote section “Blood Vessels of the Human Term Placenta and Umbilical Cord”. ILO, BH, RDP, MB, OS, SS, AS prepared and revised the Figures. AS and OP coordinated the work and compiled the manuscript. All authors contributed to revising and editing the manuscript and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Breast cancer is the most frequent neoplasm in the female population . It is aAmong the current strategies for the treatment of breast cancer, chemotherapy represents an adjuvant treatment in most subtypes of this neoplasm and a major treatment option in advanced and triple negative breast tumors ,7. TaxanThe formulation of chemotherapeutic agents into nanocarriers allows the intravenous administration of these highly liposoluble drugs without using toxic organic solvents, increases their circulation time and favors the selective location of the drug at the tumor mass, decreasing the systemic exposure to the free chemotherapeutic, and, as a consequence, reducing its adverse effects and in some cases even increasing its efficacy.The selective location of nanoformulations at tumor level can be reached by an active or passive targeting mechanism. The passive targeting of tumors is mainly attributed to the Enhanced Permeability and Retention (EPR) effect . The bloNanomedicines are being widely investigated to improve breast cancer chemotherapy, so that there are already 10 nanomedicines marketed worldwide and 21 nanoformulations under clinical research for the treatment of this type of cancer. Liposomal formulations containing paclitaxel and doxorubicin, are one of the systems most exploited for this purpose. Polymeric micelles and albumin nanoparticles and polymer conjugates have also been developed, showing, in general, a good safety profile. In fact, all approved nanoformulations of paclitaxel show higher maximum tolerated doses than free drug, indicating that its nanoencapsulation is a good strategy to overcome its administration challenges.® -albumin nanoparticles containing paclitaxel- all the nanoformulations that reached clinical stage, were based on passive targeting. However, in the last few years, the development of actively targeted nanotherapies, especially antibody-drug conjugates, has grown considerably, with two marketed formulations and a great number of them under clinical investigation. Most of them are targeted to HER-2 receptors, and, consequently, designed for the treatment of HER-2 positive tumors. However, other targets are also being investigated, such as Trop-2 receptors for the treatment of triple negative breast tumors.Initially, except in the case of Abraxane®), parenterally administered every one, three or four months, are already approved for the treatment of several hormone dependent tumors, including breast cancer.Apart from the nanoformulations, the use of micro-carriers also allows the administration of highly liposoluble antineoplastics without using organic solvents, decreasing the overall toxicity of the formulation compared to the free drug ,18. In aFinally, as we mentioned previously, the chemotherapeutic regimens include the combination of several antineoplastics. The use of nano and micro carriers for their vehiculization also allow the administration of two or more antineoplastics at the same time. This is especially interesting in the case of nanomedicines. The encapsulation of two antineoplastics in the same nanocarrier modifies their biodistribution compared with the free drugs, allowing their location at the tumor site or even inside the cancer cells at the same time, which can increase the antitumor efficacy and prevent the generation of resistances. In this way, several formulations are being investigated, including nanosystems containing paclitaxel and doxorubicin, two major antineoplastics used in breast cancer chemotherapy ,20,21,22In the present Special Issue, researchers are invited to contribute manuscripts reporting original data or updated literature reviews covering the use innovative formulations containing chemotherapeutics for the treatment of any breast cancer subtype.