Dietary patterns with intermittent energy restriction (IER) have been proposed as an attractive alternative to continuous energy restriction (CER) for the management of obesity and its associated comorbidities. The most widely studied regimens of IER comprise energy restriction on two days per week (5:2), alternate-day energy restriction by 60–70% (ADF), and timely restriction of energy intake during a specific time window within the day . Although there is some evidence to suggest that IER can exert beneficial effects on human cardiometabolic health, yet is apparently not superior compared to CER, there are still some critical issues/questions that warrant further investigation: (i) high-quality robust scientific evidence regarding the long-term effects of IER  is limited since the vast majority of intervention studies had a duration of less than 6 months; (ii) whether the positive effects of IER are independent of or actually mediated by weight loss remains elusive; (iii) it remains unknown whether IER protocols are a safe recommendation for the general population; (iv) data concerning the impact of IER on ectopic fat stores, fat-free mass, insulin resistance and metabolic flexibility are inconclusive; (v) the cost-effectiveness of IER dietary regimens has not been adequately addressed; (vi) direct head-to-head studies comparing different IER patterns with variable macronutrient composition in terms of safety and efficacy are scarce; and (vii) evidence is limited with regard to the efficacy of IER in specific populations, including males, the elderly and patients with morbid obesity and diabetes mellitus. Until more solid evidence is available, individualization and critical perspective are definitely warranted to determine which patients might benefit the most from an IER intervention, depending on their personality traits and most importantly comorbid health conditions.  Identifying effective strategies for long-term weight control is critical to reduce the alarming prevalence of obesity worldwide and mitigate obesity-associated health risks ,2. Excesper se rather than weight loss, and are thus attenuated upon cessation of the negative energy balance [The vast majority of studies on weight loss have investigated the efficacy of conventional hypocaloric diets providing continuous energy restriction (CER) ,11. Over balance . Based o balance . The mos balance ,15,16,17 balance ,9, althoThe aim of the present review is to describe the most frequently used versions of IER in clinical research settings and elucidate the health claims of this eating pattern in relation to cardiometabolic risk factor modulation. More specifically, the effects of IER on body weight, lipid profile, hypertension and glucose metabolism are presented, and a number of important concerns, limitations, knowledge gaps and unresolved questions related to IER are critically discussed. The studies reviewed herein have been performed in adults, and not in children or adolescents.As already mentioned, one of the most frequently applied patterns of IER is the alternate-day fasting (ADF) protocol, according to which energy consumption is usually reduced by 75% for three or four non-consecutive days of the week . On “freTo summarize the available evidence, it has been shown that IER can lead to a weight loss of 4–10% within a time period of 4–24 weeks, depending on the dietary pattern ,28,29,30Weight loss maintenance and prevention of weight regain represent an extremely important component of a successful weight loss intervention and depend to a great extent on the self-discipline and motivation of the dieters. Predictors of successful long-term weight maintenance after initial weight loss involve frequent self-monitoring of body weight , medicalUnder conditions of profound caloric restriction, the enhanced susceptibility to the lipolytic effects of catecholamines promotes an elevated rate of splanchnic lipolysis and an increased free fatty acid (FFA) flux . In suppWith regard to lean body mass, it is generally accepted that conventional hypocaloric diets reduce skeletal muscle mass by 10–60% of total weight loss achieved . This reStudies in overweight and obese individuals investigating the impact of IER upon glycemic profile and insulin sensitivity assessed by a variety of methods have produced heterogeneous and inconsistent findings. In some of these, IER had no effects on blood glucose and/or insulin levels in non-diabetic subjects. More analytically, no statistically significant differences were observed between IER and CER followed for 4–24 weeks, either in terms of fasting glucose ,33,61,62It has been shown that both insulin-mediated peripheral glucose disposal  and insulin-induced suppression of lipolysis (adipose tissue insulin sensitivity) during an euglycemic hyperinsulinemic clamp are significantly ameliorated after 2 weeks of IER in healthy subjects. This improvement has been mainly attributed to the increased circulating adiponectin concentrations observed after 20 h of fasting . Gender Data of comparative evaluation of IER vs. CER in terms of improving glycemic control in overweight and obese patients with T2DM are limited. In this field, it has been shown that an IER diet with caloric restriction of 70% on 4 days per week for at least 12 weeks did not result in significant alterations of HbA1c levels in overweight and obese T2DM patients compared to a continuous hypocaloric diet . SimilarThe majority of human studies have shown a beneficial effect of IER diets on the lipidemic profile of the participants ,37,38,51A recent systematic review and meta-analysis investigating the effects of IER and CER on lipid profile concluded that both dietary approaches reduce the levels of total cholesterol, LDL cholesterol and triglycerides but have no effect on HDL cholesterol . In factSeveral studies report comparable blood pressure (BP) reduction with IER and CER diets ,34,37. SA systematic review and meta-analysis investigating the effects of IER on BP levels in a total population of 1400 participants concluded that both systolic (−3.3 mm Hg) and diastolic (−1.6 mm Hg) BP can be significantly lowered with IER . It shouIER has been associated with a beneficial effect on circulating inflammatory markers in humans ,60,73. TIt has been postulated that the positive effects of IER on cardiometabolic health and beyond are partly attributed to mechanisms associated with circadian rhythms, glucose-to-ketone metabolic switching, mitochondrial function and lipid metabolism, as summarized in Sophisticated experimental studies have shown that when the master clock in the brain (suprachiasmatic nucleus), as well as other peripheral clocks  are desynchronized, the risk of cardiovascular and metabolic dysregulation is increased . In thisIER is further characterized by a metabolic switch from liver-derived glucose to adipose cell-derived ketones either daily or several days per week . PeriodsThe most important issue is that there are currently no long-term safety data with IER diets, especially in the population of normal-weight subjects.Furthermore, in normal-weight subjects who cannot adequately control the amount of ingested food (unrestrained eaters), and especially in the setting of strict dietary interventions requiring a drastic caloric restriction , IER diets have been associated with a multitude of adverse effects including increased hunger feelings, fatigue, irritability, mood disorders, concentration difficulties and uncontrolled hyperphagia on the days of unrestricted feeding, which is frequently manifested as binge eating disorder . This isThere have been also concerns that strict and prolonged hypocaloric diets, either continuous or intermittent, may disrupt the hypothalamus-pituitary-gonadal axis of young women, promoting menstrual abnormalities and potentially interfering with their reproductive ability. Although there is currently no solid evidence to suggest that IER diets have a clinically meaningful detrimental effect on ovulation and fertility of women of reproductive age , it shouAlthough there is some evidence to suggest that IER can exert beneficial effects on human cardiometabolic health, yet apparently not superior compared to CER, there are still some questions that need further examination.High-quality robust scientific evidence (based on RCTs) regarding the long-term effects of IER remains limited. The vast majority of intervention studies included in the systematic reviews and meta-analyses had a duration of less than 6 months. Of note, two RCTs of at least 12 months duration concluded that IER and CER result in similar weight loss and improve CVD risk factors to a similar extent, conveying the take-home message that IER diets (ADF and periodic fasting) are not superior to conventional CER ,79. An iWhether the positive effects of IER are independent of or actually mediated by weight loss remains elusive. In other words, it is yet unclear if IER represents another weight loss regimen or rather an independent cardioprotective and health-promoting behavioral approach. Although evidence from animal studies clearly suggests that IER itself is related to metabolic improvements , this isA critical question that needs to be answered is whether IER protocols are a safe recommendation for the general population. Possible contraindications, including psychiatric disease, food preoccupation, disordered eating and body weight regain related to the adoption of IER, are not adequately discussed in the literature .Furthermore, data concerning the impact of IER diets on ectopic fat stores, adipocyte size, fat-free mass, insulin resistance and metabolic flexibility are heterogeneous and inconclusive. Some studies in normal-weight subjects have unraveled detrimental effects of intermittent diets on fat distribution and metabolic homeostasis, which raises safety concerns and warrants further investigation. Moreover, the cost-effectiveness of IER has not been adequately addressed, and direct head-to-head studies comparing different IER patterns in terms of safety and efficacy are scarce. Evidence is also limited with regard to the efficacy of IER in specific populations, including males, children, adolescents, elderly and patients with morbid obesity and T2DM. Studies investigating the effects of IER interventions with specific characteristics in terms of macronutrient composition  are also lacking.Thus, unanswered questions in the field comprise whether long-term compliance with IER is superior to CER, whether IER-induced weight loss can be maintained for long and what the optimal characteristics are of an IER intervention  that can lead to maximal efficacy in weight loss and cardiometabolic risk factor improvement.IER is an interesting subject in the research field of body weight regulation, CVD prevention and overall health promotion. However, given the relative paucity of long-term data, it is yet not clear whether IER provides a safe recommendation for the general population. Regarding body weight loss, parameters such as long-term adherence and compliance, personalization, efficacy, high-quality foods and balanced diet, negative energy balance, realistic goal setting and safety should be underlined.The wide popularity of intermittent diets along with the knowledge gaps highlighted in the present review underscore the vital need for future rigorous research in the field of intermittent dieting with appropriately designed, long-term, randomized studies in several patient subgroups. Additional studies are also required in the population of children and adolescents who are also affected by the obesity pandemic. Until more solid evidence is available, individualization and critical perspectives are definitely warranted to determine which patients might benefit the most from an IER dietary intervention, depending on their personality traits and most importantly comorbid health conditions.


In vitro embryo production using oocytes from prepubertal cattle and buffalo collected by laparoscopy can be used to produce embryos from genetically superior females. Following transfer of these embryos into adult recipient animals, multiple offspring can be produced from these elite animals in a very short timeframe, long before they reach sexual maturity, thereby reducing the generation interval and accelerating genetic gain. This review article summarizes recent advances in this technology, outlines the current limitations, and suggests possible avenues to further improve this emerging biotechnology.Laparoscopic ovum pick-up (LOPU) coupled with in vitro embryo production (IVEP) in prepubertal cattle and buffalo accelerates genetic gain. This article reviews LOPU-IVEP technology in prepubertal Holstein Cattle and Mediterranean Water Buffalo. The recent expansion of genomic-assisted selection has renewed interest and demand for prepubertal LOPU-IVEP schemes; however, low blastocyst development rates has constrained its widespread implementation. Here, we present an overview of the current state of the technology, limitations that persist and suggest possible solutions to improve its efficiency, with a focus on gonadotropin stimulations strategies to prime oocytes prior to follicular aspiration, and IVEP procedures promoting growth factor metabolism and limiting oxidative and endoplasmic reticulum stress. In vitro embryo production (IVEP) and embryo transfer (ET) technologies have had a momentous impact on livestock production, with their use growing substantially in recent years. Despite barely being used on a commercial scale as recently as the late 1990s, IVEP has increased at an average annual rate of 12%, according to data provided by the International Embryo Transfer Society , camelidAnother key factor that explains IVEP expansion is the refinement of technologies to enable safe and practical collection of oocytes from live females. In large adult animals, most oocytes used for commercial embryo production are collected via ultrasound-guided trans-vaginal ovum pickup (OPU). However, in species that are too small for oocyte collection via OPU , a laparoscopic ovum pick-up (LOPU) procedure was developed in the early 90s . Since tOf particular interest is the application of LOPU to conduct IVEP in very young animals. LOPU allows the recovery of oocytes from animals as young as two months of age, long before they are sexually mature or large enough for ultrasound guided OPU. Subsequently, IVEP allows for these oocytes to be fertilized in vitro to produce blastocysts, which are then transferred into adult recipient females, as shown in There are two main reasons for the interest in using prepubertal animals as oocyte donors. First, the ovarian pool of available oocytes is vast; prepubertal animals consistently yield large numbers of cumulus-oocyte complexes (COCs) compared to their adult counterparts ,23. SecoBos taurus taurus) and Mediterranean water buffalo . Together, these species serve as complementary animal models to investigate prepubertal oocyte competence and improve prepubertal reproductive technologies since Holsteins mature relatively quickly while water buffalo mature much more slowly. In normal breeding practices using artificial insemination, Holstein heifers typically give birth to their first calf around two years of age, while, on average, water buffalo heifers are not expected to calf until around three years of age. As such, the goals of this review are to outline the current state of the technology, identify research gaps and suggest possible future avenues of research.This review will focus on prepubertal reproductive technologies, sometimes dubbed ‘juvenile in vitro embryo transfer’ (JIVET), in Holstein–Friesian cattle  are a family of neuropeptides in the hypothalamus, which were discovered in 2003 to operate upstream of gonadotropin-releasing hormone (GnRH) signalling [The HPO axis is essential for the management of the oestrous cycle and, consequently, fertility. Kisspeptins  and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), while decreasing levels of steroidogenic acute regulatory protein (StAR) and hydroxy-δ-5-steroid dehydrogenase, 3β-and steroid δ-isomerase 1 (HSD3B1) in calf granulosa cells . The molIn prepubertal animals, LOPU-IVEP has been performed following hormonal stimulation protocols that were adapted from those used for adult animals. The goals of gonadotropin stimulation are not only to increase the size of follicles, and consequently oocyte competence, but also to increase the number of follicles suitable for aspiration . FolliclAs the LOPU procedure is essentially the same for all ruminants and has been described in detail in other manuscripts ,10,53, tBos taurus and indicus calves [In adult cows, the ovarian response upon gonadotropin stimulation is widely variable among animals . The sams calves . This isIn the specific case of buffalo, another factor potentially contributing to variation in results is season. Buffalo are sensitive to long photoperiods, with reproductive efficiency improving in the autumn and winter as daylight decreases, similar to sheep & goat ,113,114.Following LOPU, oocytes undergo in vitro maturation, fertilization, and culture. Although variations exist in cattle and buffalo, these usually last for 22 h, 18 h, and 7 days, respectively. Most protocols have followed media compositions and procedures consistent with those used for adult animals with minimal derivations ,101,120.Although in vivo maturation was the norm for many years, and the first Holstein calf born in the world from IVF was a product of in vivo maturation , in vitrMultiple studies have shown that, although prepubertal oocytes are able to complete nuclear maturation, their ability to manage cytoplasmic maturation is more ambiguous. For nuclear maturation, it has been shown that oocytes can undergo germinal vesicle breakdown and successfully arrest at metaphase II ,47,103. The ability of calf oocytes to properly manage fertilization, oocyte activation and the block to polyspermy appears to be impaired. Research in the 1990s showed that, although fertilization rates (as measured by sperm penetration) were the same between prepubertal and adult donors, there was a significantly higher rate of abnormal fertilization in prepubertal (16%) than adult (7%) oocytes ,47. WorkFollowing fertilization, cell division appears to be delayed, with a low proportion of calf-derived embryos reaching the 4 and 8-cell stages of development at standardized time points ,120. In Recently, the possible impact of ARTs on the embryonic epigenome has garnered attention, with studies suggesting offspring produced by IVEP may be at higher risk of various disease ,127. ForDespite the lower development to the blastocyst stage, prepubertal embryos can reach this stage in a similar timeframe and have normal characteristics including a visible inner cell mass . In termThe ultimate and essential test for blastocyst quality is the ability to establish pregnancy and result in healthy offspring following embryo transfer. Pregnancies and live births with full-term offspring following LOPU-IVEP and embryo transfer have been reported by multiple authors using calf-derived oocytes in both Holsteins ,53,101 aIn addition to yielding similar rates of embryos and pregnancies following transfer, another goal is for prepubertal-derived embryos to have cryotolerance similar to that of adult-derived embryos. It is well documented that in vivo produced embryos are more cryotolerant than their in vitro produced counterparts ,136,137.With the knowledge that prepubertal LOPU-IVEP technologies do work, as evidenced by healthy calves born following embryo transfer, the current challenge is improving efficiency. As such, attention should focus on conditions both in vivo, before LOPU, and in vitro, following LOPU. In vivo approaches should include innovative gonadotropin stimulation protocols for young donor animals in order to enhance the intra-follicular environment and maximize oocyte development inside the follicle. In vitro approaches should focus on amending IVEP procedures to better accommodate the requirements of prepubertal oocytes to maximize meiotic maturation, normal fertilization and embryo development to the blastocyst stage.Efficient gonadotropin stimulation regimes should increase the size of follicles available for aspiration, as embryo development rates are directly associated with follicular size ,72,92,96Oxidative stress caused by reactive oxygen species (ROS) can damage cells by disrupting homeostasis and leading to apoptosis. Glutathione (GSH) is considered the major line of defence against oxidative injury by helping to maintain the redox state within the cell. In addition to its role in preventing oxidative stress, GSH has been shown to play an important role in the transport of amino acids, as well as in DNA and protein synthesis . The triAlthough many different antioxidants have been tested and used over the years in adult IVEP schemes, there are fewer studies assessing the efficacy in prepubertal animals, especially in cattle and buffalo. Working with 1–2-month-old goats, Rodriguez-Gonzalez and colleagues found that IVM supplemented with cysteamine increased the GSH concentration, and improved blastocyst yield and total cell number per blastocyst . SimilarAnother antioxidant used in many IVEP schemes across multiple species is melatonin, which has been shown to reduce oxidative damage in the oocyte ,156. MelEndoplasmic reticulum (ER) stress is a major contributor to embryonic death because physiological and exogenous stressors typically lead to disruptions in protein folding and ROS production in the ER . InductiCytokines and growth factors are small peptide proteins involved in cellular signalling and communication. Fibroblast growth factor 2 (FGF2), leukaemia inhibitory factor (LIF), and insulin-like growth factor (IGF1) are among the growth factors found in follicular fluid that have regulatory effects on COCs. Working with porcine oocytes, Yuan and colleagues (2017) assessed the impact of adding these growth factors (in a cocktail coined ‘FLI’) to IVM media and observed a significant increase in oocyte maturation, embryo development and quality, and litter size following embryo transfer . WorkingThere is evidence that a short ‘pre-maturation’ period in presence of meiotic inhibitors such as c-type natriuretic peptide (CNP), epidermal growth factor receptor (EGFR) inhibitor, and cAMP prior to IVM may improve oocyte competence. During LOPU, separation of COCs from their follicles causes cAMP concentrations to decrease, resulting in spontaneous resumption of meiosis . During Although several obstacles remain to be overcome, the use of prepubertal breeding schemes based on LOPU-IVEP is a powerful method for accelerating genetic gain. In Holsteins, the technology has reached a level of commercial viability, with several large biotechnology companies currently using this technology. Although the potential reward in applying this technology in buffalo is larger due to their prolonged sexual maturity, more work needs to be done for further efficiency optimization. Enhanced stimulation protocols yielding more competent oocytes at collection, coupled with in vitro procedures that will improve cytoplasmic maturation and the oocyte’s machinery to fight oxidative and ER stress, are among the improvements that will likely increase the proportion of competent oocytes recovered from prepubertal compared with post-pubertal animals.


Glioblastoma multiforme is the most lethal intrinsic brain tumor. Even with the existing treatment regimen of surgery, radiation, and chemotherapy, the median survival time is only 15–23 months. The invasive nature of this tumor makes its complete removal very difficult, leading to a high recurrence rate of over 90%. Drug delivery to glioblastoma is challenging because of the molecular and cellular heterogeneity of the tumor, its infiltrative nature, and the blood–brain barrier. Understanding the critical characteristics that restrict drug delivery to the tumor is necessary to develop platforms for the enhanced delivery of effective treatments. In this review, we address the impact of tumor invasion, the molecular and cellular heterogeneity of the tumor, and the blood–brain barrier on the delivery and distribution of drugs using potential therapeutic delivery options such as convection-enhanced delivery, controlled release systems, nanomaterial systems, peptide-based systems, and focused ultrasound. Glioblastoma multiforme (GBM) is the most common primary brain malignancy in adults, accounting for more than 50% of intrinsic brain tumors . AccordiWidely recognized risk factors associated with GBM occurrence are exposure to high-dose ionizing radiation and certain genetic syndromes, including neurofibromatosis type 1 and Li-Fraumeni syndrome ,9,10. ThGBM is characterized by rapid cell proliferation and extensive invasion of tumor cells into the surrounding brain, making complete removal of the tumor impossible . These fIn this review, we discuss the impact that molecular and cellular tumor heterogeneity, tumor dispersion and the blood–brain barrier have on the delivery and distribution of pharmacological agents to GBM. The currently available chemotherapeutic delivery systems designed to overcome these constraints are also discussed . We willGBM is characterized by molecular heterogeneity within a single tumor in addition to inter-patient heterogeneity. Molecular heterogeneity underlies the cellular heterogeneity in GBM, i.e., the differences between cell types within a tumor . This moIn addition to the molecular heterogeneity described above, GBM cells are morphologically and functionally heterogeneous. Single-cell RNA-Seq studies of individual tumor cells have revealed that GBM tumor cells exist in multiple stages of differentiation . Other sThe blood–brain barrier is composed of a highly specialized circuit of blood vessels that are lined by brain microvascular endothelial cells (BMEC), the cell–cell junctions between which restrict the entry of potentially harmful substances  into the brain. The BMECs are surrounded by pericytes, astrocytes and the basal membrane ,23. ThisDisruption of the blood–brain barrier (BBB) is observed in glioma tumor regions. Unfortunately, this degree of BBB disruption is not sufficient to allow for the ability of therapeutics to reach this diffusely infiltrating tumor ,27. BiocIn this review, we will discuss the application of controlled release systems, convection-enhanced delivery, nanomaterial systems, peptide-based therapeutics, and focused ultrasound for drug delivery to glioblastoma, overcoming the obstacles posed by GBM molecular and cellular heterogeneity, GBM cell invasion/dispersal and the blood–brain barrier.Implantable drug release systems enable the direct delivery of therapeutic agents to the tumor site, circumventing the need to cross the blood–brain barrier. Drug-loaded biocompatible materials such as drug-impregnated gels can be designed so that they release low doses of drugs at the tumor site over a prolonged period of time.® wafer. This is a biodegradable polymer wafer loaded with the chemotherapeutic drug BCNU. It is the only FDA-approved drug delivery implant for treating GBM [Implantable controlled-release delivery systems can be constructed with degradable or non-degradable polymers. Between these two options, biodegradable polymers are more commonly used . One sucting GBM . Gliadelting GBM ,32,33. Iting GBM . Biodegrting GBM . The rigting GBM .A pH-responsive carboxymethylcellulose biopolymer system was used to deliver the chemotherapeutic drug, rhodamine B encapsulated in multiple emulsions to glioblastoma cells in vitro . In a moWith sustainable release of therapeutic agents over time, implantable controlled-release systems limit the local GBM recurrence after resection by interacting with the tumor cells at the resection site. However, these systems are not only limited by a variety of side effects as listed above, but also by poor drug distribution to distant tumor cells that have migrated in the normal brain parenchyma. Consequently, these implantable local delivery systems have limited ability to affect infiltrating GBM cells distant to the site of implantation.Convection-enhanced delivery (CED) is a catheter-based drug delivery method that depends on pressure gradients rather than diffusion to deliver therapeutic agents into brain tumors. This technique involves the stereotactic insertion of one or more catheters into the tumor. The catheters are connected to a syringe/drug delivery pump that maintains a positive pressure gradient that promotes the distribution of higher volumes of drug over a larger area. Ensuring direct intratumoral drug delivery independent of drug concentration or diffusivity of the infusate, CED allows the use of large volumes of the drug at lower and less-toxic doses . The ratCED of temozolomide, the standard cytostatic drug for the treatment of GBM, combined with subcutaneous immunizations with irradiated cancer cells had a synergistic effect on tumor growth and overall survival in a mouse-GL261 glioma model. However, the same protocol did not display synergy in the KR158 mouse glioma model, which is resistant to radiation and chemotherapy. In the latter model, CED of temozolomide increased survival . By initIn a phase I clinical trial, CED was used to infuse carboplatin to patients with WHO grade III astrocytomas or grade IV gliomas (GBM). This approach increased median overall survival without any systemic toxicity . This waMRI-guided CED of iron oxide nanoparticles (IONP) conjugated to epidermal growth factor receptor deletion mutant III antibody (EGFRVIIIAb) showed significant increase in survival in a mouse GBM model . BindingDirect delivery of anticancer agents in large volumes and at less toxic doses makes CED an attractive drug delivery method for GBM. Implantable catheters can be used to repeat CED at required intervals, but the efficacy of this approach may be limited by the highly invasive nature of the tumor with GBM cells dispersed to distant sites . ChallenNanomedicine is an emerging candidate for cancer therapy that uses a variety of different types of nanocarriers such as lipid-based, polymer-based, inorganic, viral, and drug-conjugated nanoparticles . Small sNanomaterial systems for drug delivery improve GBM tumor targeting mainly by promoting drug diffusion through the blood–brain barrier and by the enhanced permeability and retention (EPR) effect. The EPR effect is dependent on the highly angiogenic nature of GBM where leaky vasculature is commonly present . BecauseThe delivery of tumor antigens and adjuvants using nanoparticles as a vehicle has been shown to increase the efficacy of immune therapy against GBM ,56,57. DA nanobubble-based theranostic system consisting of intravenously administered iron-platinum nanoparticles loaded with doxorubicin and surface-functionalized with transferrin  reduced glioma growth in a mouse model by almost 70%. The nanobubbles were burst by exposing to high intensity focused ultrasound (HIFU) to bypass the blood–brain barrier by generating a cavitation effect. This single nanocomposite, combining nanomaterials, chemotherapeutic agents and MRI contrast agents all together for the first time, was able to cross the blood–brain barrier, target the tumor cells and allow imaging of the brain tumor. This multimodal system is an example of combining several strategies to enhance the success rate of therapy .The physicochemical properties of nanomaterials enable them to deliver therapeutic agents to the brain via drug encapsulation or by surface modification. The clinical use of the nanomaterial drug delivery systems is limited by the poorly controlled accumulation and distribution of particles in and around the specific target site ,62. The There are three main types of peptide-based therapeutics—tumor homing peptides, peptides targeting aberrant cellular signaling pathways and cell-penetrating peptides . GBM celHighly selective tumor-targeting peptides obtained using a biopanning phage display library directed against GBM cells were able to cross the BBB and deliver the oncolytic virus VSVΔM51 to GBM in a mouse model in vivo . These pSelf-assembled spherical nanoparticles containing a peptide probe (Cy5.5-SAPD-99mTc) that combines tumor homing ability with mitochondria targeting was found to have promising theranostic possibilities owing to the enhanced apoptosis in GBM cells coupled with imaging functionality . IncorpoPeptide derivatives of rabies virus glycoproteins, RVG29 and RVG15-liposome, were used to deliver anticancer chemotherapeutic docetaxel nanoparticles and paclitaxel-cholesterol to glioma-bearing ICR mice with a positive effect on animal survival ,71. The WSW  is a tumor-targeting peptide (derived from Clostridium acetobutylicum) that was fused to paclitaxel nanosuspensions and used to target GBM cell membranes in a glioma mouse model. The use of WSW induced apoptosis and prolonged survival of the animals . By combThe use of polydopamine (PDA)-coated zein-curcumin nanoparticles functionalized with the peptide G23 inhibited cell proliferation and migration in glioma cells in vitro . Here, tA dual peptide nanocomplex created by combining SynB3 (a cell penetration peptide) with PVGLIG (an MMP-2 sensitive peptide) and paclitaxel inhibited cell migration and invasion in multiple GBM cell lines, suppressed GBM tumor growth in vivo, and increased overall survival in a mouse model of GBM . The abeTo enhance the membrane permeability of peptides, membrane receptors such as low-density lipoprotein receptor, IL-4 receptor, and transferrin receptor which are abundantly expressed on GBM cells have been used to direct the delivery of tumor-homing peptides to brain malignancies, utilizing receptor-mediated transcytosis ,79,80. PFocused ultrasound (FUS) is an image-guided, noninvasive method to transiently open the blood barrier, thereby enhancing the efficacy of therapeutic delivery to GBM. FUS can be used to reversibly disrupt the BBB without irreversible tissue damage . The useBy increasing BBB permeability, FUS can enhance the delivery of varied therapeutic agents to the tumor. For example, FUS-induced disruption of the BBB enhanced the local delivery of temozolomide to tumors and increased the overall survival of rats harboring experimentally induced gliomas . FUS appThe use of imaging techniques along with FUS increases the rate of drug delivery to specifically identified tumor tissues. MRI guided FUS (MRgFUS) was used to achieve a higher tissue delivery of liposome-encapsulated doxorubicin in rats, temozolomide in mice, and cisplatin-conjugated gold nanoparticles in mice ,88,89. IUsing this noninvasive technique, relatively low systemic doses of therapeutic agents can be used, thereby reducing systemic toxic effects . TransieMolecular and cellular heterogeneity, GBM cell dispersal and the BBB are critical constraints limiting the efficacy of anti-GBM drug therapy. Applications of CED, controlled-release systems, nanomaterial systems, peptide-based therapeutics and focused ultrasound for drug delivery to tumor enhance survival with reduced toxicity in animal studies . Despite


Generally, a large tumor size of hepatocellular carcinoma (HCC) is associated with poor visibility and uncertainty in the surgical field which results in increased surgical difficulty as well as unfavorable postoperative outcomes. We performed intended preoperative trans-arterial embolization (TAE) in patients with a large HCC. In this study, we investigated the oncological significance of intended preoperative TAE for a large HCC, using a comparison between patients with and without TAE, and detailed analyses for pre- and post-TAE status.n=51) and those with smaller HCC  according to the size of their HCCs. Comparison of clinicopathological features between these groups and clinical outcomes between the TAE and non-TAE groups were compared. In addition, a detailed analysis of each case in the TAE group was conducted, comparing clinicopathological factors between pre- and post-TAE status.A total of 411 patients who underwent hepatectomy for primary HCC at the University of Yamanashi Hospital between January 2007 and December 2018 were included in this study. The patients were divided into two groups: patients with larger HCCs  were revealed compared to those with small HCCs (<50 mm). The prognostic analyses showed that a large tumor size and increased tumor markers, multiple tumor numbers, and others were adverse prognostic factors, and vascular invasions and residual tumors were included in the multivariate analysis.Further detailed analyses revealed that the average rates of change in tumor size and tumor shrinkage after TAE were − 48.6±35.6 mm and − 30.7±17.0%, respectively. Pathological high necrotic changes in the tumor, after multiple-times TAE aiming to a better effect, were related to a better prognosis in patients with large HCC. Poor prognostic factors became less common in patients who underwent intended preoperative TAE, and these patients had better prognoses.The large tumor size of HCC is associated with unfavorable outcomes; the intended preoperative TAE for large HCC patients performed multiple times aiming to affect the tumor as much as possible might improve their prognoses. Liver cancer is one of the major malignancies and ranks 2020) sixth in incidence and third in mortality worldwide ; hepatoc020 sixthGenerally, a large tumor is associated with poor visibility and uncertainty in the surgical field which results in increased surgical difficulty . In addiA total of 411 patients who underwent hepatectomy for primary HCC at the University of Yamanashi Hospital between January 2007 and December 2018 were included in this study. Patients with non-HCC tumors  were excluded. This study was approved by the Ethics Committee of the University of Yamanashi  and was performed in accordance with the ethical standards of the Declaration of Helsinki and its later amendments .The reserved liver functions and blood examination, including tumor markers such as alpha-fetoprotein (AFP), the L3 fraction of AFP (AFP-L3), and protein induced by vitamin K absence or antagonist II (PIVKA-II), were indicated as pre-treatment (pre-TAE and preoperative) data. Reserved liver functions were evaluated using the Child-Pugh classification and indocyanine green (ICG) retention test. Hepatitis was classified as HBV, HCV, NBNC including alcoholic hepatitis and NASH, and others. Tumor sizes were evaluated based on pathological findings of resected tumors, while preoperative tumor size was measured using computed tomography (CT) imaging. Clinical data, including laboratory examination results, postoperative prognoses, and operative outcomes, such as intraoperative blood loss (IBL) and operative time, were obtained from the electronic medical recording system at the University of Yamanashi Hospital.After evaluation of general and tumor statuses using blood tests and imaging examinations, huge HCCs were revealed in several patients; these were perceived by hepatology surgeons as considerably difficult surgical procedures with a risk of a large amount of IBL and/or deficient volume of the remnant liver. Therefore, these patients underwent the intended preoperative TAE for the purpose of tumor shrinkage and of facilitating difficult surgical procedures because of the large tumor size. TAE manipulation was performed by professional radiologists using conventional methods with lipiodol and/or antitumoral agents. The therapeutic effects of TAE were evaluated using contrast CT examination and blood tests. TAE procedures were practiced repeatedly to achieve a sufficient effect against tumor viability. In addition, with or without preoperative TAE, some patients who were assumed to undergo extended liver resection were pretreated with portal vein embolization. After these intended preoperative procedures, surgical resections were performed for patients with large HCCs.Postoperative follow-up was performed with evaluation of tumor markers every 3 months and imaging examination by contrast CT or magnetic resonance imaging and/or complementary abdominal ultrasonography every 3 to 6 months . Recurren=51) and group S  according to the size of their HCCs. The patients’ background, including age, sex, reserved liver function and hepatitis etiologies, and tumor-associated factors such as tumor markers, tumor number (single or multiple), status of vascular invasions, and perioperative outcomes, were compared between the two groups. Furthermore, univariate and accompanied multivariate prognostic analyses for 5-year DSS were performed.To evaluate the significance of preoperative TAE, three steps of analysis were conducted in this study. First, the patients were divided into two groups: group L . Among them, five patients had undergone preoperative TAE aimed at tumor shrinkage and/or oncological effect (TAE group). Comparative analysis between patients in the TAE group and those without preoperative TAE (non-TAE group), particularly focusing on the aforementioned prognostic factors in the first prognostic analysis, and prognostic analyses for 2-year RFS along with 5-year DSS were performed. Finally, a detailed analysis of each case in the TAE group was conducted. Changes in tumor size, laboratory examination, relationship between frequency of TAE, pathological necrosis rate, and prognosis were evaluated.t-test. Univariate prognostic analyses were performed using the Kaplan–Meier method and log-rank test. A multivariate prognostic analysis was conducted using factors that were indicated with a p value of less than 0.1 according to the results of the univariate analysis. All statistical analyses were performed using EZR , a graphical user interface for R  [p<0.05.All continuous variables have been represented as median values and their ranges or mean ± standard errors, unless noted otherwise. Statistical analyses were performed using the chi-square test and Student’s Austria) . StatistThe results of the comparison for all patients in groups L and S are shown in Table p=0.049) and 5-year DSS , respectively. However, the results also showed that there was still a certain level of difference in tumor sizes between the two groups even after the intended preoperative TAE. Due to such differences, patients in the TAE group underwent longer operations which were associated with higher IBL and blood transfusion; this was also demonstrated as a poor prognostic factor in the previous analysis.Comparison of characteristics between the two groups (TAE and non-TAE groups) demonstrated that patients in the TAE group had no residual tumor and had relatively lower tumor markers which were indicated as prognostic factors in the aforementioned univariate and multivariate prognostic analyses Table . In factTable We noted clinically unfavorable short- and long-term outcomes in patients with large HCCs (≥50 mm) compared to those with small HCCs (<50 mm). Prognostic analyses showed that a large tumor size and increased tumor markers and multiple tumor numbers were adverse prognostic factors; vascular invasions and residual tumors were then included in the multivariate analysis. Further detailed analyses revealed that these factors became less common in patients who underwent intended preoperative TAE and had better prognoses. Thus, we have provided the oncological significance of the intended preoperative TAE for patients with large HCCs.For gastrointestinal cancers including HCC, as well as other malignancies, both surgical resection and perioperative treatments have been more important along with improved treatment effects –15, and HCCs show different recurrence patterns compared to other gastrointestinal malignancies—HCC is more likely to show intrahepatic metastases than systemic distant metastases, and intrahepatic recurrences are divided into two detailed patterns. One is multi-centric occurrence based on chronic hepatitis, and the other is intrahepatic metastatic recurrence from the primary HCC which mainly occurs through the intrahepatic portal veins . In factThe prognostic significance of preoperative TAE has been well investigated in the 1990s and the 2000s. Some studies have suggested a favorable effect for patients with HCCs; however, most researchers did not agree with the positive results and concluded that preoperative TAE was not performed uniformly. One reasonable explanation for these unfavorable results might be the activation of tumor cells due to hypo-oxygenic stimulation caused by the insufficient therapeutic effects of TAE. In fact, Sasaki et al. suggested that preoperative TAE should be avoided for these patients, especially those without cirrhosis or with an early-stage tumor; however, most of the patients included in the study underwent only a single embolization . In contThis study had some limitations. First, it was difficult to derive definitive conclusions due to the small number of patients who underwent intended preoperative TAE in this study; further prospective and large-scale trials are necessary to address this. Additionally, there might be some biases in the indication of preoperative supplemental treatment; this depends on the surgeons’ decision and the strategies of TAE followed by the radiologists. Furthermore, advanced HCC has various statuses to be discussed other than tumor size, such as lymph node metastasis , and cliIn conclusion, the large tumor size in HCC is associated with unfavorable outcomes and intended preoperative TAE for large HCC patients (performed multiple times) might improve their prognoses.


Preterm infants are known to have long-term healthcare needs. With advances in neonatal medical care, younger and more preterm infants are surviving, placing a subset of the general population at risk of long-term healthcare needs. Oral feeding problems in this population often play a substantial yet under-appreciated role. Oral feeding competency in preterm infants is deemed an essential requirement for hospital discharge. Despite achieving discharge readiness, feeding problems persist into childhood and can have a residual impact into adulthood. The early diagnosis and management of feeding problems are essential requisites to mitigate any potential long-term challenges in preterm-born adults. This review provides an overview of the physiology of swallowing and oral feeding skills, disruptions to oral feeding in preterm infants, the outcomes of preterm infants with feeding problems, and an algorithmic approach to the evaluation and management of neonatal feeding problems.  Occurring in over 10% of live births in the world annually, prematurity is considered a significant global healthcare problem . In the Insults during organogenesis and tissue differentiation can impact the developing fetus or preterm infant, resulting in short-term and long-term implications. Developmental plasticity helps the fetus develop a phenotype best suited to the environment, but these short-term adaptations could result in the future development of pathological states . Barker Oral feeding readiness in preterm infants is a concern often towards the tail-end of hospitalization. This only comes into the spotlight after other major medical concerns have been resolved or are manageable. While the evidence for the adult outcomes of preterm infants is increasing, the long-term impact of the altered development of oral neuromotor skills and abnormal oral feeding remains unclear, primarily due to a paucity of studies on adults born preterm. Given the diversity of underlying etiologies and clinical presentations of feeding problems, the lack of standardized definitions and assessments by which to identify and document oral feeding problems adequately is frustrating . There iIn this review, we will appraise (1) the physiology of swallowing and oral feeding skills, (2) disruptions to oral feeding in preterm infants, (3) feeding problems in preterm infants beyond the NICU, and (4) an algorithmic approach to the evaluation and management of neonatal feeding problems. Normal swallowing begins with the formation of a food bolus in the mouth and ends with the bolus being carried down into the stomach through a series of coordinated functions involving sucking, swallowing, respiratory endurance, and airway protection with overarching neurological control. Anatomical changes such as decreasing relative tongue size and lower laryngeal position with growth play an important role in the evolution of the swallowing mechanisms . The precursors to oral feeding skills emerge in the early second trimester of pregnancy with evidence of sucking and swallowing in the developing fetus. Early evidence of fetal swallowing is seen at 11–12 weeks of gestation by the swallowing of amniotic fluid, followed by evidence of early sucking at 18–20 weeks ,11. As t—the oral phase, the pharyngeal phase, and the esophageal phase [Mature swallowing occurs broadly in three phasesal phase ,16. The al phase ,17,18,19al phase .As the food bolus is pushed to the back of the oral cavity, a largely involuntary swallowing reflex is triggered in the pharyngeal phase. This leads to a sequence of events from the elevation of the velum leading to nasopharyngeal closure, followed by the pharyngeal swallowing reflex with the contraction of superior, middle, and inferior constrictors . A brief respiratory pause with the closure of the vocal cords from the glottic closure reflex and laryngeal chemoreflexes help protect the airway during this phase. As the bolus moves to the esophageal phase, this activates a series of actions from hyoid elevation, relaxation of the cricopharyngeal muscle, opening of the esophagus, and the eventual peristaltic contraction of the esophagus  relaxation, primary peristalsis of the esophageal body, and lower esophageal sphincter (LES) relaxation) that moves the food bolus into the stomach, thus concluding the swallow .The phrase “feeding problems” is often used to describe a diverse group of etiologies resulting in inadequate nutritional intake orally and could range from oral feeding difficulties (described in more detail below), gastroesophageal reflux (GER), dysmotility, and even systemic illnesses, unfortunately contributing to the lack of a uniform definition for feeding problems. The maturation of a normal feeding pattern can be interrupted in a preterm infant by extrauterine influences resulting in dysphagia or abnormal swallowing, with impaired sucking, swallowing, or airway protection, thus compromising the efficacy, adequacy, and, most importantly, the safety of oral feeding. Dysphagia can be a sign of underlying anatomical or neurological abnormality or injury, or merely functional immaturity altered by external sensory input, as seen in a preterm infant . InfantsPreterm infants, especially those born <32 weeks, are exposed to multiple life-saving interventions during their NICU stay that contribute to noxious orofacial sensory stimulation during a time of physiologic maturation. Additionally, during such a vital period of brain development, prolonged oxygen supplementation due to respiratory illnesses (respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD)), brain injury (intraventricular hemorrhage (IVH), periventricular leukomalacia (PVL) or hypoxic injury), complex medical illnesses such as necrotizing enterocolitis (NEC), surgeries, and underlying craniofacial abnormalities can further alter oral sensory and motor experiences impacting normal sucking and swallowing development. There exists a complex interplay between prematurity, pulmonary, neurological, and feeding problems independently linked to neurodevelopmental delays. Oral phase disruptions: The oral phase is altered in preterm infants who experience prolonged external stimulation . Immature or absent oral reflexes result in subtle to overt symptoms from an abnormal oral phase, including weak and disorganized sucking, immature patterns such as biting or chewing, and poor bolus formation and propulsion, leading to dribbling during feeding ,16. BreaPharyngeal phase disruptions: The swallowing mechanism is intricately linked to respiratory efficiency. The pharynx is the only common pathway between the airway and digestive tract during swallowing and requires a coordinated response between swallowing and breathing for successful feeding . ImmaturEsophageal phase disruptions: The pharyngeal reflexive swallow and pharyngo-lower esophageal sphincter reflex were previously shown by Jadcherla and colleagues to be immature and underdeveloped in preterm infants compared to term infants . AdditioAspiration during swallowing can occur broadly in three scenarios—anterograde aspiration in the oropharyngeal phase as bolus travels into the pharynx, retrograde aspiration (refluxed food bolus travels up the esophagus with failure to protect the airway), and silent aspiration . Additionally, an abnormal pharyngoglottal closure reflex and esophagoglottal reflex associated with prematurity also contribute to abnormal airway protective mechanisms. Oral feeding and non-invasive respiratory support: It is not uncommon for preterm infants who are starting to develop oral feeding skills at 33–34 weeks postmenstrual age to continue to require respiratory support due to lung disease (RDS or BPD). Oral feeding such preterm infants on moderate respiratory support such as nasal CPAP or high-flow nasal canula (HFNC) has been debated over the years. Some studies have shown that oral feeding on CPAP may be associated with increased risk of aspiration ,23, whilThere is currently a dearth of large-scale studies describing feeding problems in children and adults born preterm, and the descriptions of feeding problems vary significantly between previous studies with a high degree of heterogeneity. In a large NICHD neonatal research network (NRN) study, Adams-Chapman et al. defined abnormal feeding as the need for gastrostomy or tube feeds, choking, gagging, excessive drooling, or difficulty swallowing during oral feeds, physicians’ orders to not feed by mouth, and confirmed aspiration . ConversFeeding problems in childhood and adults among preterm born infants have been described inconsistently. While previous studies have suggested that up to 1% of the pediatric population can present with feeding problems, the prevalence is likely to be significantly higher in preterm infants, proportional to the degree of prematurity . ResiduaAs the feeding skills mature into childhood with more voluntary control, it is only plausible that feeding problems become more apparent in infants with neurological injury and impaired cortical function. In a recently published meta-analysis by Pados et al., including 22 studies, the prevalence of feeding problems was 42% in the first four years of life amongst preterm-born infants . In a suIn a retrospective cohort of 194 preterm infants of <37 weeks with feeding difficulties referred to a specialized feeding disorders program, 40% of infants were discharged on gastrostomy tube (GT) feeds, and out of these, the majority (78%) were still receiving GT feeds at one year of age, either full GT (40%) or partial GT feeds (38%) . These GPostnatal malnutrition associated with feeding problems can result in failure to thrive (FTT) and developmental delays. Postnatal nutritional compromise can modify adult outcomes in these children resulting in multisystem abnormalities as an adult. In the NICHD study mentioned above, Warren et al. also showed a high incidence of FTT amongst the infants discharged home on GT feeds. Out of the 333 ELBW infants (7.3%) that were discharged on GT, 56% had FTT at an 18–22-month follow-up ,37,38,39Oral aversion was described in a population survey conducted in the UK at two years corrected age comparing 1130 preterm versus 1255 term infants. The authors identified an increased incidence of feeding difficulties, picky eating/refusal, and oral motor problems in the preterm infant cohort compared to those born term. The adjusted relative risk for total feeding problems was 1.57, refusal/picky eating was 1.53, and oral motor problems was 1.62. According to a multivariate analysis, the authors noted that prolonged nasogastric tube >2 weeks was independently associated with increased eating difficulties . Speech Gastrointestinal reflux is a frequent indication for the use of thickened feeds and anti-acid medications in preterm infants. A population based study based on the Swedish birth registry showed an inverse relation between gestational age and the use of anti-acid medications per year for gastroesophageal reflux disease in adulthood . Of all Eosinophilic esophagitis (EoE) is chronic immune-mediated inflammatory disease of the esophagus characterized by eosinophilic inflammation of the esophagus. Multiple case–control studies in both pediatrics and adults have shown that prematurity is one of the early-life risk factors for developing eosinophilic esophagitis in one’s later years of life. In a case–control study comparing children with EoE versus healthy controls, premature infants had 46% higher odds of developing EoE compared to term infants, with an adjusted odds ratio of 1.46. Multiple pediatric and adult studies have shown a variable odds ratio of 1.46 to 2.92 for prematurity being an early-life risk factor for developing EoE in childhood or adulthood ,43,44.Feeding problems are described in multiple genetic syndromes associated with hypotonia in infancy. Term infants with trisomy 21, even in the absence of major anomalies (40%), have more feeding problems when compared to their term counterparts (34% vs. 15%) without trisomy 21 . InfantsCongenital heart diseases have long been linked to feeding problems even after several years following cardiac surgery, leading to a substantial number of infants needing nasogastric tubes or GT for nutrition ,50. FeedMaternal metabolic conditions such as hypertension, diabetes, and drug use have been linked to feeding problems in neonates. Infants of diabetic mothers have an increased prevalence of feeding issues during the early neonatal period. There is increasing evidence that feeding and eating disorders persist in infancy and early childhood. A population-based cohort study from Denmark showed a 64% increased risk of feeding and eating disorders in children of mothers with diabetes . NeonateIntrauterine growth restriction (IUGR) is linked to nutritional compromise in a fetus, preventing it from reaching its full genetic potential, and it is principally a vascular disorder with short-term and long-term consequences ,57,58. TPreterm infants are also prone to higher rates of anoxic brain injury and IVH, with increased incidence of cerebral palsy often manifesting as subtle dysphagia. While the data on the long-term outcomes of preterm infants discharged home on tube feedings are sparse, term infants with congenital cardiac disease who are discharged home on an artificial mode of providing nutrition such as orogastric tubes or GT have poorer mental and psychomotor developmental indices . It woulFeeding problems are dynamic and continue to evolve as the infant grows, requiring an ongoing assessment, intervention, and re-evaluation to optimize management. Early diagnosis and treatment of feeding problems involve a holistic approach to the individual’s specific feeding difficulties in order to decrease potential long-term challenges ,19. FiguA detailed history includes prenatal history, maternal medical problems and exposures, birth history, neonatal course by systems, and a description of the signs and symptoms. Physical examination should evaluate the anatomy of the oropharyngeal structures and identify craniofacial anomalies and neurological status, including cranial nerves. The quality and maturity of the sucking, milk expression, and sucking–swallowing coordination are assessed. Abnormalities during a bedside feeding, such as wet voice, gagging, choking, and drooling, could suggest a possible airway compromise that may require targeted instrumental assessment. However, it is essential to note that the pediatric population is suspected of having a high prevalence of silent aspiration of unknown significance and may not be identified accurately on clinical assessments alone. Several bedside tools are now available for oral motor assessment of infant feeding skills. Some commonly used tools in the neonatal period include Infant Driven Feeding Scales (IDFS), Neonatal Oral Motor Assessment Scale (NOMAS), the Oral Feeding Skills Scale (OFS), and newer tools such as the Neonatal Eating Assessment Tool (NeoEAT) and Neonatal Feeding Assessment Scale (NFAS) ,61,62,63Multidisciplinary feeding teams including neonatology, speech pathology, and occupational and physical therapy are integral to understanding the intricacies of feeding issues related to prematurity and the complex interplay of other comorbidities. Neonatal interventions for dysphagia are primarily conservative, involving non-pharmacological management. Feeding readiness assessments for maturity and respiratory safety—informal or structured—are essential to guide the initiation of oral feeds in a preterm infant. Oral motor therapy is an essential element for the improvement of readiness to start feeding ,17. The Bolus modifications such as thickened feeds may be used in some cases as the infant becomes closer to term gestation ,67. In aInstrumental assessments can be used to evaluate the anatomy or function of aerodigestive structures and the presence of GER as the infant grows . A VFSS Both VFSS and FEES are complementary studies that can provide different perspectives. Studies comparing VFSS and FEES showed significant agreement between the two ,76. An upH-multichannel intraluminal impedance (pH-MII) is a combination diagnostic tool used to evaluate the esophageal phase of GER, including pH changes that can diagnose the presence and frequency of gastroesophageal reflux episodes . pH-MII Pharyngo-esophageal high-resolution manometry (PE-HRM) is an advanced tool used to understand abnormal aerodigestive motility reflexes and help guide management. In a cohort of 74 preterm infants with GER symptoms evaluated by both pH impedance and provocative esophageal manometry, a higher correlation of GER symptoms was noted with increasing stimulus volumes used for esophageal provocation, highlighting the role of esophageal peristaltic reflexes . In anotPharmacological interventions are often the last resort in the neonatal period and are frequently limited to treating GER symptoms. The role of anti-acids is limited and recommended to treat GER only after optimizing non-pharmacological measures, including thickening feeds, modifying feeding volume and frequency, and positional therapy . The AmeMotility agents such as erythromycin have been used in severe cases of feeding intolerance with dysmotility. Erythromycin is not recommended in preterm infants <32 weeks , while iFeeding problems encompass diverse interlinked etiologies often grouped together. There is an urgent need to subcategorize feeding problems based on underlying etiology to provide targeted diagnostic and therapeutic support. The diagnosis of dysphagia is limited by available techniques and expertise, and, when diagnosed, there are few reliable treatment options in this delicate population. Parents and caregivers are often frustrated with a lack of a quick fix for feeding problems in infancy. Nevertheless, with patience and perseverance, many interventions show results in due time with delayed gratification. A multidisciplinary team approach to feeding in the NICU with established algorithms for early oral motor stimulation, initiation, and advancement of oral feeds using quality improvement methodologies is vital for the mitigation of future feeding problems . FurtherAdditionally, as more preterm infants survive into adulthood, the long-term effects and healthcare costs of feeding problems are also expected to increase tremendously in the coming decades . Standar


Unlike animal studies, hMPS models can be configured for high content or high throughput screening in preclinical drug development. Applications in modeling acute and chronic injuries in the musculoskeletal system are slowly developing. However, the complexity and load bearing nature of musculoskeletal tissues and joints present unique challenges related to our limited understanding of disease mechanisms and the lack of consensus biomarkers to guide biological therapy development. With emphasis on examples of modeling musculoskeletal tissues, joints on chips, and organoids, this review highlights current trends of microphysiological systems technology. The review surveys state-of-the-art design and fabrication considerations inspired by lessons from bioreactors and biological variables emphasizing the role of induced pluripotent stem cells and genetic engineering in creating isogenic, patient-specific multicellular hMPS. The major challenges in modeling musculoskeletal tissues using hMPS chips are identified, including incorporating biological barriers, simulating joint compartments and heterogenous tissue interfaces, simulating immune interactions and inflammatory factors, simulating effects of in vivo loading, recording nociceptors responses as surrogates for pain outcomes, modeling the dynamic injury and healing responses by monitoring secreted proteins in real time, and creating arrayed formats for robotic high throughput screens. Overcoming these barriers will revolutionize musculoskeletal research by enabling physiologically relevant, predictive models of human tissues and joint diseases to accelerate and de-risk therapeutic discovery and translation to the clinic.Human Microphysiological Systems (hMPS), otherwise known as organ- and tissue-on-a-chip models, are an emerging technology with the potential to replace  Musculoskeletal conditions encompass a wide spectrum of pain or damage associated with muscle, bone, cartilage, tendon, ligament, joints, and nerves. Injuries to musculoskeletal tissues are the leading cause of disability worldwide and often limit mobility and restrict the patients’ ability to work or participate in recreational activities. As the mean age of the population increases, it is expected that the prevalence of musculoskeletal conditions and the associated socioeconomic burden will increase drastically in the coming decades . The mosThe process for drug and therapeutic discovery, development, and approval is arduous and costly. Discovery typically starts in academic or pharmaceutical laboratories. Unfortunately, many discoveries in academic laboratories fail to be reproducible or scaled up in pharmaceutical research and development laboratories. Leading drug candidates may be abandoned in the developmental “valley of death” because they are too slow or too costly to develop. And even well-funded efforts can fail in clinical trials despite promising preclinical findings. As a result, only ∼10% of the therapeutic development pipelines entering phase I clinical trials typically proceed to FDA approval. Without a technological breakthrough that reduces risk and front-end investments required for drug development, these significant barriers will continue to hamper development and translation efforts .Less than 8% of active interventional clinical trials of musculoskeletal diseases in the United States involve disease-modifying biological therapies, including stem cell therapy, growth factors and platelet-rich-plasma trials. The shortage of biological therapies for musculoskeletal conditions, excluding arthritis, highlights several issues for the field to recognize and tackle. The American Academy of Orthopedic Surgeons (AAOS) Symposium in 2015 identified the root cause as the incomplete knowledge of musculoskeletal disease mechanisms and a lack of reliable biomarkers to inform clinical trials . In addiin vitro culture systems is constrained by the limitations of these models. Animal models have been the cornerstone of translatable biomedical research over the past century. Despite their undeniable value in biomedical research, animal models have numerous limitations that unfortunately have contributed to the arduous and costly new therapy development process. Animal studies are intrinsically low throughput and do not accurately predict the drug’s effects and bioavailability in humans due to differences in pharmacokinetic and pharmacodynamic (PK/PD) responses. In addition, animal models used for biomedical studies are almost always inbred for research purposes and thus lack the genetic diversity of the human population. Lastly, studies of experimentally-induced acute or chronic musculoskeletal conditions do not use consensus models to allow uniformity in outcomes and valid interpretations of different experiments. Current in vitro culture models represent artificial, non-physiological conditions, mostly consisting of a single cell type or at best co-cultures of two cell types, to simulate the paracrine signaling between immune cells  and mesenchymal cells . Three dimensional (3D) scaffolds such as collagen are often used, but they are typically monocellular and not readily amenable to modeling the heterogeneity in a tissue. These systems are, therefore, inadequate to faithfully model treatment effects on acute or chronic musculoskeletal conditions or predict clinical outcomes.The level of evidence obtained from preclinical studies using animal models and in vitro systems provide relevant, accessible, and flexible models of tissues and organs. MPS typically use microfluidic channels or compartments that model micro-scale units of multicellular tissues or organs  from adult somatic cells , CRISPR/r organs , tissue r organs , and mulr organs . Ideallyr organs . The intr organs . Therefor organs . Such woin vivo mechanical loading, incorporating vascularization and innervation, and recreating joints and sophisticated soft-to-hard tissue interfaces.Despite the proliferation of these sophisticated chips to model various tissue and organ systems, scientists in the musculoskeletal field have been slow to develop and adopt them. For example, there has been significant progress in developing and validating MPS for major diseases associated with high mortality rates such as the heart , lung H, intestiTherefore, this review outlines current tools and trends in the MPS field, citing examples of early applications to model musculoskeletal diseases. In addition to providing an overview of critical issues in MPS, the review discusses specific challenges that should be prioritized in future MPS models of musculoskeletal diseases to accelerate their adoption into the drug and therapeutic discovery pipeline and in virtual clinical trials.in vitro before demonstrating efficacy in TMJ reconstruction studies in large animals , microdevices engineered to contain patterned cells and ECM to model tissue and organ structure and function at the micro-scale. The defining characteristics of OoaC include recreating the 3D arrangement of tissues on the platform, patterning multiple organotypic cells in a physiologically relevant context, and simulating biochemical  and biophysical (forces) cues relevant to the modeled tissue or organ. For example, the effectiveness of co-culturing 3D human skeletal muscle fiber tissues with human iPSC-derived motor neurons to study the NMJ depends on whether the model is 2D or 3D. When 3D NMJ-on-chip models were compared to 2D culture conditions, they demonstrated functional superiority in fiber maturation and Acetylcholine receptors (AChR) clustering that affected the electrophysiological responses, strongly suggesting that 3D NMJ-on-chip is a powerful model to study adult human sensorimotor synaptogenesis and NMJ disease in vitro   dia flow . The BM-dia flow . AdditioThere are various cell sources for MPS, which include primary cells, cell lines, stem cells, and iPSCs. Each source has advantages and disadvantages, which must be factored in their utilization in MPS applications. Arguably, these cells must be derived from human sources for the MPS to have their highest impact.in vitro  in culture before they reach senescence and lose their ability to divide. This loss of proliferative ability is attributed to reduced telomerase activity at high passage numbers . Therefoin vitro . These dStem cells are perhaps the single most important discovery in regenerative medicine. They possess properties that can theoretically correct pathological changes caused by disease or injury. However, stem cells and their progenies can also be used as primary components of personalized (patient specific) MPS models of human disease. By definition, stem cells are characterized by the ability to self-renew indefinitely by symmetric or asymmetric cell division while maintaining an undifferentiated state and the ability to differentiate into the various fates of specialized cell types under the right chemical and biological cues. This latter property refers to the regenerative potency of stem cells. While the nomenclature is generally not uniformly used, pluripotent stem cells can differentiate into cells from any of the three embryonic germ layers: the ectoderm, the mesoderm, and the endoderm. First successfully isolated from the inner cell mass of the blastocyst by James Thompson in 1998 , human eAlternative sources of stem cells include extraembryonic fetal tissues such as the placenta and the umbilical cord (Wharton’s jelly). In addition, stem cells have been identified in specialized compartments or niches in numerous tissues that retain a moderate level of regenerative abilities throughout postnatal and adult life. These adult stem cells are multipotent, such that they can differentiate into different cell types that make the tissue or related tissues from the same embryonic germ layers. As with primary cells, the invasive isolation of human adult stem cells is associated with tissue injury and donor site morbidity that constrain their applications in MPS models.in vitro  and mesenchymal stromal  cells (MSC), is a replenishable, readily accessible source of adult stem cells with minimal morbidity. HSC give rise to myeloid and lymphoid progenies of blood and immune cells through well-characterized steps of differentiation, which can be replicated in vitro . As such in vivo . Caplan  in vivo . Protoco in vivo . Numerou in vivo ]. Stem c in vivo . And whi in vivo , their uOct3/4, Klf4, Sox2, and c-Myc pluripotency factors   . Today,  testing .In particular, the use of human iPSC-derived cells from an autologous donor overcomes the previously intractable problem of creating interconnected, isogenic MPS models of multiple tissues or organ, paving the way towards clinically relevant human-on-chip models . TherefoDespite their undeniable potential for MPS applications, the use of human iPSC in modeling human diseases has limitations. The relationship between the genome and epigenome has broadened the understanding of the types of molecular events that cause human disease. Current strategies for iPSC generation/regeneration of isogenic tissues eliminates this epigenetic memory from donor cells while maintaining the patient’s intact genome. Although human iPSC are effective for modeling purely genetic disease , they have limitations in modeling diseases stemming from both genetic and epigenetic factors. They also have other challenges in modeling important biological variables that maybe systemic rather than purely cellular, including sex and age.As with stem cells, the utility of human iPSCs in MPS models of musculoskeletal diseases requires efficient and reproducible multilineage differentiation protocols. Strategies to generate human iPSC musculoskeletal derivatives have been described in numerous publications, including osteoblasts , tenocytPolymeric hydrogels are widely used in MPS platforms as they resemble the macromolecular ECM of many tissues and organs, providing proper cellular architecture, support, and function. The three main categories of hydrogels include natural, synthetic, and hybrid materials. Animal sourced natural hydrogels such as collagen and fibrin are biocompatible and provide native cell-binding ligands and biochemical properties present in native tissues. On the other hand, the process of deriving natural hydrogels from animals results in limited mechanical strengths, long-term storage instabilities, and batch differences, reviewed in . Alternain vivo and biomechanically matching the tissue of interest with a biomaterial scaffold. Although not thoroughly covered here, others have extensively reviewed the biomaterials and fabrication methods which are key components in successful representation of musculoskeletal disease models , synthetic, or hybrid. The primary advantages of these approaches are the controlled architecture acquired through scaffolding and high-definition fabrication techniques achieved with electrospinning and 3D-printing. Limitations of these approaches include the difficulty in recreating cell-ECM interactions e models .Beyond the conceptual design of the MPS, practical considerations influence the choice of materials used in device fabrication. The most commonly used fabrication materials for MPS devices are elastomers and rigid polymers. The choice of the fabrication materials affects manufacturability, assembly flexibility, maintaining sterility, incorporating precise physical stimuli , molecular adsorption and absorption, and longitudinal monitoring of outcomes through live microscopy or integrated sensors.Elastomers are cross-linked polymers with weakly entangled chains. Due to their low elastic moduli and weak intermolecular forces, they easily deform (stretch or compress) and experience high strains without failure when an external force is exerted but return to their undeformed state when the force is withdrawn. Polydimethylsiloxane (PDMS) is an elastomer that has been widely adopted in the microfluidics community for its versatility, biocompatibility, permeability, and low cost. PDMS has been crucial in the early work of MPS and remains a prodigious material, with most devices still utilizing PDMS as their primary structural component. Despite its popularity, some limitations of PDMS include incompatibility with inorganic solvents, absorption of small hydrophobic molecules, adsorption of biomolecules, and gas permeability that lead to changes in concentrations of solutions over time . The mulRigid thermoplastic polymers such as Polystyrene (PS), Poly (methyl methacrylate) (PMMA), Polyurethane, Teflon, and PEGDA are high strength, relatively inflexible, low cost, light weight, optically transparent, and biocompatible (low monomer leaching) materials widely used in fabricating MPS devices. These rigid thermoplastics can be reshaped multiple times by reheating, which is advantageous for molding and bonding. These materials can be fabricated through silicon master molds, reactive ion etching (REI), injection molding and hot embossing. Therefore, thermoplastic components of MPS devices can have high upfront production and development costs not feasible for prototyping but suitable for large batch manufacturing. As an alternative, rapid prototyping methods include CNC micromilling techniques and 3D printing to allow for quick, low-cost fabrication at the benchtop.2 exchange is necessary for buffering the cell media and can accumulate in impermeable, static platforms. Yet, it can be optimal when using media with premixed gases to monitor dissolved oxygen consumption and pH levels in the MPS environment for example  model the disease within the context of the tissue’s physiological and functional parameters, 2) enable longitudinal and endpoint assays, and 3) accommodate the skills, resources, and objectives of the end user. Physiological and functional parameters include gradients, heterogenous interfaces, biological barriers, mechanical or electrical stimulation, fluid flow, and interconnectivity with different tissue or organ chips. Longitudinal assays include brightfield and fluorescent microscopy, multiplex sensing of secreted proteins, while destructive endpoint assays include histology, immunohistochemistry, and q-PCR. End users could be trainees in academic laboratories pursuing high content data to uncover mechanisms of disease, drug discovery scientists in a pharmaceutical R&D facility pursuing high throughput data to identify hits that could be developed as drugs or biologics, or clinical trial technicians seeking proof of safety or efficacy of a drug candidate.in vivo loading, and inflammation. Importantly, engineering outcomes of pain indicators in MPS is critical to designing clinical trials-on-chip since pain is one of the most significant patient-reported outcomes in the clinic.In addition to the challenges of creating clinically-faithful disease models with biomarkers that capture the dynamic nature of acute or chronic pathologies, tissues and joints in the musculoskeletal system, there are various specialized features that require engineering innovations to model them on MPS. For example, the migration of innate and adaptive immune cells from the bone marrow into the vasculature, the infiltration of platelets, neutrophils, macrophages, and various immune cells to sites of tissue injury, and cancer metastases growth underscore the importance of engineering permeable vascular barriers. Biological interfaces and ECM gradients, such as the myotendinous junction and the enthesis are critical not only for mechanical function but also for cellular functions and signaling, and there are several engineering approaches to engineer gradients that would need to be scaled down for MPS. Directed motor neuron terminal attachment to highly organized muscle fibers is another example of the intricate microarchitectural engineering that would be required to model innervated tissues . FurtherThe following sections discuss seven challenges that should be prioritized in future musculoskeletal MPS platforms to increase the predictive power of these models in disease research and drug discovery areas. These challenges include engineering biological barriers, engineering heterogenous tissue interfaces, incorporation of immune cells and inflammatory factors, biomechanical actuation and loading, incorporating surrogate measurements for pain, integrating inline sensors for real-time monitoring of dynamic processes, and creating arrayed formats for high throughput screening.The following section describes compartmentalized approaches used to engineer biological and vascular barriers in MPS with application examples in musculoskeletal disease models .in vivo transporting epithelium  in a methacrylated gelatin hydrogel. The cell-laden construct was placed in a bioreactor with two separated fluidic channels accessing the top and bottom of the construct respectively. A chondrogenic medium was perfused in the top channel and an osteogenic medium was supplied through the bottom conduit over 28 days to induce chondrogenic/osteogenic differentiation of the iMPCs. The construct was shown to effectively model OA in the cartilage compartment through the introduction of IL-1β, and responds by reducing inflammatory cytokines when treated with Celecoxib, a COX-2 inhibitor commonly used as a first-line treatment for OA .Directly patterning microchannels into the MPS substrate is an effective approach to control the cellular architecture and organization. Microchannels link two adjacent chambers and can be lined with monolayer forming cells such as endothelial or epithelial cells to completely cover the inner surfaces or they can be used to guide axonal growth. Microchannels have been used to model the human neuromuscular junction (NMJ) transmission upon exposure to inhibitors, where motoneurons (MNs) can communicate with skeletal muscle cells in two separate compartments connected by microchannels embedded in a PDMS BioMEM construct. Physiological behavior was evidenced in the system as high frequency excitation of the MNs drove the myotubes to contract into tetanus while pharmacological NMJ inhibitors added to the muscle compartment demonstrated that MN-induced muscle contraction could be attenuated . Such a in vivo vasculature, such as in the bone marrow, a substantial challenge in musculoskeletal tissues that must be overcome to develop physiologically relevant MPS models.Perfusable channels are often designed to be embedded in hydrogel constructs within MPS, usually to form vascular networks. Vascular networks are critical to many diseases such as metastatic cancer, inflammation, and fibrosis. Incorporating vasculature into musculoskeletal MPS also permits immune cell components to be introduced and drug studies to be tested through vascularized models which can provide estimates of PK/PD. Vascular networks for MPS have been successfully incorporated into various tissues and organs and showed improved outcomes for disease modeling and physiological function . VasculaAnother approach to address this challenge is the self-assembled formation of vasculature from endothelial lined microchannels through a gel-liquid interface with angiogenic gradients driving vascular growth in the hydrogel . These min vitro such as the cartilage-bone junction, neuromuscular junctions, myotendinous (muscle-to-bone) junction, and entheses (tendon-to-bone junction) into MPS are significant unmet needs. Modeling injury to biological junctions in a humanized model requires the integration of multiple tissues and phases into one MPS. This can be a particularly challenging process in vitro where cell matrix needs differ widely. In musculoskeletal tissues, the integration of soft-to-hard interfaces Inflammation represents the body’s response to cell and tissue damage from harmful agents or injury. The initiation, progression, and resolution phases of inflammation in musculoskeletal tissues and disease are important considerations in developing effective treatments. Acute injury to musculoskeletal tissues initiates the recruitment of inflammatory cells  to the injury site. Neutrophils, the first phagosomes recruited, set the stage to the activation of monocytes and lymphocytes to attack and eliminate foreign organisms and agents. The functional consequences of activation of circulating monocytes to macrophages, which represent the innate immune response, depend on their polarization, with M1 macrophages effecting phagocytosis and proinflammatory cytokines secretion and M2 macrophages typically credited with anti-inflammatory cytokine and growth factor secretion to initiate tissue repair . Lymphocin vivo. For example, the intrinsic vs. extrinsic factors affecting the fate, heterogeneity and plasticity of synovial macrophages is still largely unknown, yet breakthroughs in this area can dramatically improve joint treatments  for phenotypic drug testing has been developed . The fabonotecan . Such mon models . This trin vitro human models, also known as tissue- and organ-on-a-chip, carefully designed to offer standardized predictive models by mimicking physiologically relevant aspects of living tissues and organ systems. Applications in modeling musculoskeletal acute and chronic injury are slowly developing. Keys to translational implementation MPS models of musculoskeletal pathologies include developing strategies to: engineer vascular and biological barriers and heterogenous tissue interfaces; incorporate immune cells and inflammatory factors; enable biomechanical actuation to simulate in vivo loading; incorporate sensory neurons (nociceptors) to record surrogate measurements for pain; integrate inline sensors for real-time monitoring of secreted proteins critical to modulating these dynamic processes; and develop arrayed formats for high throughput screening. Furthermore, issues of scalability, reproducibility, and validation, which are not discussed in this review, are also of paramount importance and have been addressed in other reviews (The poor translation from preclinical animal studies to human clinical applications and incomplete understanding of the mechanisms of action and the lack of biomarkers to define biological efficacy represent significant barriers that impede the development of disease modifying therapies for musculoskeletal conditions. The emerging technology of hMPS might offer transformative opportunities to cost-effectively address the aforementioned barriers. MPS is a wide-encompassing term for sophisticated  reviews .