Relapse is a major obstacle to curb the ongoing epidemic of prescription opioid abuse. We and others previously demonstrated that oxycodone seeking in adult rats progressively increases after abstinence from oxycodone self-administration (incubation of oxycodone craving). In humans, the onset of oxycodone use in adolescents may increase individuals’ vulnerability to later opioid addiction. However, little is known about incubation of oxycodone craving after adolescent-onset oxycodone self-administration in rats. In the first study, we trained single-housed adolescent  and adult  male Sprague–Dawley rats to self-administer oxycodone  and then tested oxycodone relapse on both abstinence day 1 and day 15. Given that social experience is critical for neurobehavioral development in adolescents, we performed the second study using group-housed adolescent and adult rats. In both studies, we observed no age differences in oxycodone self-administration and incubated oxycodone seeking on abstinence day 15. However, on abstinence day 1, we observed decreased oxycodone seeking in adolescents compared with adults. This pattern of data led to elevated incubation slopes in adolescent rats compared with adult rats. Finally, group-housed rats exhibited attenuated oxycodone seeking compared with single-housed rats on abstinence day 15, but not on day 1. Taken together, these data suggest that adolescents may be resistant to oxycodone relapse during early abstinence, but this resistance dissipates quickly during the transition between adolescent and young adulthood. In addition, group-housing plays a protective role against incubated oxycodone craving. The opioid epidemic is an ongoing public health crisis, affecting millions of people across the United States. In the past 20 years, over 200,000 overdose-related deaths involved prescription opioids, such as oxycodone . One of In humans, the onset of drug use, including opioids, often occurs during the adolescent period and young adulthood . Early-oHowever, it is unknown whether adolescent-onset oxycodone self-administration impacts relapse behavior in the adulthood after abstinence. Note that previous studies on the effect of adolescent-onset drug self-administration on drug seeking yielded mixed results  see det. We founn = 62) or 63 . Adolescent rats were at P 35 and adult rats were at P 77 at the beginning of the self-administration training. In Experiment 1, we group-housed rats 4 per cage before surgery and then housed them individually after surgery (single-housed condition). In Experiment 2, rats were group-housed four per cage before and after surgery (group-housed condition). In the case that we lost cage mates due to health-related issues, we ensured that there were no less than three rats per cage in the group-housed condition. All rats were maintained under a reverse 12:12-h light/dark cycle with food and water freely available. We performed the experiments under the protocols approved by the University of Maryland College Park Animal Care and Use committee. We excluded rats due to catheter patency , poor training , and health-related issues . The number of rats reported herein refers to rats included in the statistical analysis.Male Sprague–Dawley rats (Charles River) arrived in our animal facility at either postnatal day (P) 21  and inserted silastic catheters into the rat’s jugular vein, as previously described . We injevia polyethylene-50 tubing, protected by a metal spring. We then attached the liquid swivel to a 20-ml syringe via polyethylene-50 tubing and to a 22-gauge modified needle . To promote lever pressing for adolescents, we set the active lever 3 cm lower during the first 4 days of training.We trained the rats in self-administration chambers located inside sound-attenuating cabinets and controlled by a Med Associates  system. Each chamber has two levers located 8–9 cm above the floor. During self-administration training, presses on the retractable (active) lever activated the infusion pump (which delivered an oxycodone infusion); presses on the stationary (inactive) lever were not reinforced with the drug. For oxycodone intravenous infusions, we connected each rat’s catheter to a liquid swivel (Instech) We used a training procedure previously described by The daily training sessions started at the onset of the dark cycle and began with the extension of the active lever and the illumination of the red house light. The house light remained on for the duration of the 6-h session. During training, active lever presses led to the delivery of an oxycodone infusion and a compound 5-s tone-light cue (the tone and light modules were located above the active lever). During the 20-s timeout, we recorded the non-reinforced lever presses. We set 90 infusions as the maximum for each 6-h session to prevent overdose. The red house light was turned off and the active lever retracted after the rats received the maximum infusions or at the end of the 6-h session.During the abstinence phase, we housed the rats either individually or in groups in the animal facility and handled them two to three times per week.We conducted all relapse tests immediately after the onset of the dark cycle. The sessions began with the extension of the active lever and the illumination of the red house light, which remained on for the duration of the session. Active lever presses during testing  resulted in contingent presentations of the tone-light cue, previously paired with oxycodone infusions, but did not result in drug infusions.All rats first received intravenous surgeries. Next, we trained them to self-administer oxycodone for 10 days. On abstinence day 1, we tested the rats for relapse in a 30-min session. On abstinence day 15, we tested the rats for relapse in a 60-min session. Note that we used a 30-min session on abstinence day 1 to minimize the effect of extinction learning that could decrease drug seeking on abstinence day 15 . We perft-test as appropriate. We followed significant interaction or main effects with Fisher PLSD tests. For the repeated measures analyses of the training data, we replaced 24 outlier values of inactive lever presses with the group mean for a given training day. We defined outliers as three median absolute deviations (MADs) above the group median  mixed ANOVAs or p median , and we The goal of Experiment 1 was to examine whether adolescent-onset oxycodone self-administration leads to changes in oxycodone seeking after abstinence compared with adult-onset oxycodone self-administration under the single-housing condition. To achieve this goal, we used a within-subject design and tested rats for oxycodone seeking (relapse tests) on abstinence day 1 and 15 .F9,405 = 12.378, p < 0.001), but no significant effect of Age or interaction between these two factors , but no effects of Age or interaction among these three factors  and within-subject factor of Training day (1–10). We observed a significant effect of Training day . Post hoc analysis of active lever presses revealed that oxycodone seeking was significantly higher on abstinence day 15 than day 1 in both age groups . We also observed a significant interaction between Day and Age . Post hoc analysis of active lever presses revealed that on abstinence day 1, but not day 15, there was a significant decrease in adolescent rats, compared with adult rats . In addition, the analysis of the time course data on the active lever included the between-subject factor of Age (adult and adolescent) and the within-subject factor of Session Minutes . We observed a significant effect of Session Minutes  and Age , but no interaction between these two factors on abstinence day 1 , but no effect of Age or interaction between these two factors.In both groups, oxycodone seeking was significantly higher on abstinence day 15 than on day 1. However, oxycodone seeking in the adolescent group was lower than the adult group on abstinence day 1, while no age differences in oxycodone seeking were observed on abstinence day 15. As rats were tested for relapse in a 30-min session on abstinence day 1 and a 60-min session on abstinence day 15, we analyzed the data in the following two ways. First, we performed the analysis using the data from the day 1 relapse test, and the first 30 min of the day 15 relapse test. This analysis included the between-subject factor of Age (adult and adolescent), and the within-subject factor of Abstinence day (1 and 15) and Lever (active lever and inactive lever). First, we found no interaction among these three factors , but no significant effects of Age or interaction between these two factors , and the within-subject factor of Session Minutes  and Lever (active lever and inactive lever). We found main effects of Session Minutes , but no significant effect of Age or interaction between these two factors , but no effects of Age or interaction among these three factors . Post hoc analysis of active lever presses revealed that oxycodone seeking was significantly higher on abstinence day 15 than on day 1 in both age groups . Furthermore, while the analysis of total lever presses revealed no interactions between Day and Age or a significant main effect of Age  and Session Minutes , with no interaction between these two factors , but no effect of Age or interaction between these two factors. Finally, the analysis of the 60-min relapse test on abstinence day 15 showed main effects of Session Minutes , but no significant effects of Age or interaction between these two factors , while no statistical significance was detected under group-housing condition (p > 0.05). In addition, there was a trend toward significant main effect of Housing . These results suggest that single-housed adolescent rats may have an accelerated incubation compared with single-housed adult rats, which may be interfered under the group-housing condition.In both experiments described above, we observed lower non-incubated oxycodone seeking in adolescents than adults, but no differences in incubated oxycodone seeking. Based on this observation, we are interested in comparing the development of incubation, indexed by incubation slope, following adult-onset and adolescent-onset oxycodone self-administration. We analyzed the incubation slope using the between-subject factors of Age and Housing. We found no interaction between these two factors, but a significant main effect of Age (p > 0.05). We analyzed the active lever data from the 30-min relapse test on abstinence day 1, or from the 60-min relapse test on abstinence day 15, using the between-subject factors of Housing condition (single-housing and group-housing) and Age (adult and adolescent). On abstinence day 1, we observed main effects of Age , but no effects of Housing condition or interaction between these two factors , but no effects of Age or interaction between these two factors , while there was a trend toward a decrease in oxycodone seeking in group-housed compared with single-housed adult rats .Based on substantial evidence demonstrating that environmental enrichment such as social housing attenuates drug seeking across different drug classes , we wereTaken together, these analyses demonstrated that group-housing condition attenuated oxycodone seeking on abstinence day 15, but not on day 1. This attenuating effect of group-housing on oxycodone seeking is more robust in adolescents than adults.In this study, we compared incubation of oxycodone craving following adult-onset and adolescent-onset oxycodone self-administration in single-housed and group-housed rats. Our main finding is that there were no age differences in incubated oxycodone seeking on abstinence day 15, but decreased oxycodone seeking in adolescents compared with adults on abstinence day 1, in both single-housed and group-housed rats. This pattern of data led to the finding that under the single-housing condition, adolescent rats exhibited elevated incubation slopes compared with adult rats. Regarding the effect of housing conditions, we found that on abstinence day 1, the group-housing condition had no effect on oxycodone seeking in both age groups. In contrast, on abstinence day 15, the group-housing condition in adolescent rats significantly attenuated oxycodone seeking compared with the single-housing condition, while in adult rats, the suppressing effect of group-housing was modest. Finally, we observed no age or housing-condition differences in oxycodone self-administration. Overall, these data demonstrate that adolescent rats may be resistant to relapse during early abstinence, but this resistance dissipates quickly once rats reach young adulthood. Additionally, the effect of group-housing was selective for oxycodone seeking after prolonged abstinence, but not during early abstinence.In both studies, we found no age differences in oxycodone self-administration in rats at a dose of 0.1 mg/kg under extended access condition (6 h/day). We chose this experimental condition based on previous studies from us and others detecting reliable oxycodone self-administration and incubation of oxycodone craving in adult rats . In contUnder single-housing condition, we found no age differences in incubated oxycodone seeking on abstinence day 15, which is in line with the previous finding showing no age differences in incubation of cocaine craving following adult-onset and adolescent-onset cocaine self-administration in rats . In contThe third potential factor is the presence of cues during relapse tests. An unexpected finding here is that while no age differences were observed regarding total oxycodone intake, adolescent rats exhibited attenuated oxycodone seeking on abstinence day 1, compared with adult rats. This result suggests that adolescents might be resistant to oxycodone seeking during early abstinence after oxycodone self-administration, which contrasts with no age differences in oxycodone seeking observed on abstinence day 15. A key difference between these two time points is that on abstinence day 1, the adolescent group was still in adolescent period, but they reached young adulthood on abstinence day 15 . Therefopost hoc analysis identified the significant differences of the incubation slope between ages only under the single-housing condition, but not under the group-housing condition. Together with the trending significant main effect of Housing (p = 0.083), it is possible that the suppressing effect of the housing condition on oxycodone seeking on abstinence day 15 (see below) interferes with the development of incubation.Finally, taking data from abstinence days 1 and 15 together, we identified an elevated incubation slope in adolescent-onset rats, compared with adult-onset rats, suggesting that adolescent-onset rats accelerated the development of incubation during abstinence. Therefore, one possibility is that adolescent-onset rats might exhibit enhanced oxycodone seeking than adult-onset rats in relapse tests beyond abstinence day 15. Although we cannot exclude this possibility, we believe this is unlikely because incubated oxycodone seeking in adult rats plateaus on abstinence day 15, without future increase on abstinence day 30 . AlternaFirst, we observed no main effect of housing conditions on oxycodone self-administration, which is in line with a recent study showing no effect of environmental enrichment on heroin self-administration in adult rats . In contpost hoc analysis identified the significant differences of oxycodone seeking on abstinence day 15 between housing conditions only in adolescent rats, with a trend toward statistical significance in adult rats (p = 0.090). The modest effect in adult rats may be due to the timing of housing manipulation, as discussed in detail below.On abstinence day 15, we found that group-housing attenuated oxycodone seeking. Several studies previously demonstrated that environmental enrichment, using a combination of group-housing and enrichment apparatuses in home cages, decreases cue-induced seeking to cocaine , 2012, mOn abstinence day 1, we found no main effect of housing condition. The lack of statistical significance in adolescent rats could be due to a floor effect attributed by low lever responding on abstinence day 1. In adult rats, our finding is inconsistent with previous studies showing that environmental enrichment with a combination of social housing and enrichment apparatuses in home cages decreases cue-induced cocaine seeking  and sucrThe main finding in our study is that in male rats, adolescent-onset oxycodone self-administration, compared with adult-onset, led to decreased oxycodone seeking during early abstinence, but oxycodone seeking was similar after prolonged abstinence. This result further invalidates the notion that adolescent drug exposure enhances vulnerability to relapse in adulthood, which is in contrast with the enhanced rewarding effect of opioids observed in previous studies . InsteadOne question for future studies is to assess the effect of age and housing on the severity of oxycodone withdrawal during abstinence. Regarding the age, previous studies primarily focusing on morphine and heroin suggest that exposure to opioid during adolescent leads to decreased opioid withdrawal severity, which could be reversed by re-exposure during adulthood . In contThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the University of Maryland College Park Animal Care and Use committee.XL conceived the project, provided intellectual inputs, and wrote the manuscript. RA carried out experiments, analyzed the data, and wrote the manuscript. KG carried out experiments and analyzed the data. All authors contributed to the article and approved the submitted 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 novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Mesenchymal stem cells (MSCs) are currently utilized in clinics for pulmonary inflammatory diseases, including acute respiratory distress syndrome and acute lung injury. Given that MSCs offer a promising treatment against COVID-19, they are being used against COVID-19 in more than 70 clinical trials with promising findings. Genetically engineered MSCs offer promising therapeutic options in pulmonary diseases. However, their potential has not been explored yet. In this review, we provide perspectives on the functionally modified MSCs that can be developed and harnessed for COVID-19 therapy. Options to manage the SARS-CoV-2 infection and its variants using various bioengineering tools to increase the therapeutic efficacy of MSCs are highlighted. Despite the vaccines developed thus far against SARS-CoV-2, vaccine studies continue to be developed against variants that arise due to the rapid mutation of the virus . A completed phase 1 clinical trial indicated that multiple intravenous infusions of MSCs do not adversely affect the course of COVID-19 disease and contributed to the decrease in all inflammatory cytokines within 14 days (NCT04252118). In addition, an open label dose escalation phase 1 trial demonstrated that infusion of 400 million umbilical cord-derived MSCs showed no dose-limiting toxicity [According to the clinical trial database  are no longer detectable in the body [All active clinical trials registered on the body . A phaseclinicaltrials.gov on the administration of MSC secretome and extracellular vesicles in the treatment of COVID-19 . Secretome is defined as the set of bioactive factors, such as enzymes, soluble regulatory proteins and growth factors, secreted by a specific type of cell [Besides MSC infusion, there are 7 active clinical trials registered on  of cell . The mai of cell . An MSC- of cell . In line of cell .In addition to the therapeutic effects MSCs offer, the possible risks of MSC transplantation should be considered to create a more reliable and complete view. In general, the potential risks of the clinical administration of MSCs comprise a pro-coagulant profile of MSCs ,145, uneMSC-based products express variable levels of highly procoagulant tissue factor (TF/CD142), creating a safety concern for the IV infusion of MSCs . To prevTaken together, proposing genetically modified MSCs may provide an improved MSC-based therapy for COVID-19. A potential concern in clinical settings over reducing the pro-inflammatory properties and/or enhancing the anti-inflammatory properties of MSCs may be the excessive suppression of the innate immune system. In this case, the immunomodulating effects of MSCs can be achieved by constructing gene regulatory systems. For instance, inducible systems can be used to manage the initiation and termination of the signal necessary to control the immunomodulation.Even though it is hard to predict how the engineered MSCs will respond to all these regulatory dynamics, it is much more about hope rather than hype when considering current attempts of engineered MSC therapy in several diseases.The recent preclinical data and phase trials suggest MSCs as potential therapy vehicles for COVID-19 cases. Following a brief discussion on possible engineering scenarios, here we suggest a prospective solution for the continuous and controllable secretion of anti-inflammatory cytokines by modified MSCs which may then enhance the therapy against SARS-CoV-2. This moldable approach mainly targets SARS-CoV-2-based deadly infection. The strategy can also be versatile in managing altered cytokine storms caused by the new probable variants. Further studies will provide a better understanding to assess the efficiency of our proposed hypothesis.


Osteoarthritis (OA) has traditionally been known as a “wear and tear” disease, which is mainly characterized by the degradation of articular cartilage and changes in the subchondral bone. Despite the fact that OA is often thought of as a degenerative disease, the catabolic products of the cartilage matrix often promote inflammation by activating immune cells. Current OA treatment focuses on symptomatic treatment, with a primary focus on pain management, which does not promote cartilage regeneration or attenuate joint inflammation. Since articular cartilage have no ability to regenerate, thus regeneration of the tissue is one of the key targets of modern treatments for OA. Cell-based therapies are among the new therapeutic strategies for OA. Mesenchymal stem cells (MSCs) have been extensively researched as potential therapeutic agents in cell-based therapy of OA due to their ability to differentiate into chondrocytes and their immunomodulatory properties that can facilitate cartilage repair and regeneration. In this review, we emphasized current knowledge and future perspectives on the use of MSCs by targeting their regeneration potential and immunomodulatory effects in the treatment of OA. Osteoarthritis (OA) is the most common form of degenerative joint disease, typified by degeneration of cartilage and osseous overgrowth. The incidence of OA occurs in OA affects 13.9% of individuals aged 25 and older, and 33.6% of those over the age of 65 [OA is characterized by the breakdown of joint cartilage and subchondral bones. Articular cartilage is an essential structural component of the human body. It is composed of specialized cells known as chondrocytes. These chondrocytes generate a substantial number of extracellular matrix (ECM), which is made up of collagen fibers, elastin fibers and proteoglycan. Under physiological conditions, this matrix undergoes a constant remodeling process where both degradative and synthetic enzymes known as matrix metalloproteinases (MMPs) are tightly regulated . These MIn OA, the MMPs such as collagenases, stromelysins and gelatinases are overexpressed, changing the balance in favor of net degradation and resulting in the loss of collagen and proteoglycan from the matrix . SubsequCurrent OA therapeutic approaches focus on pain relief and symptom control rather than treating or slowing progression of the disease. Hence, there is an urgent need to explore new therapeutic strategies for OA. Cell-based therapies which involve the delivery of mesenchymal stem cells (MSCs) to the osteoarthritic joint have emerged as a promising strategy to the current treatment by targeting their regeneration potential and immunomodulatory effects. In this review, we discuss the current status of stem cell therapies, and also recent advancements and future perspectives in MSC therapy for OA.OA is often regarded as a degenerative disease in contrast to rheumatoid arthritis; however, recently the degenerative theory is no longer acceptable due to increasing evidence suggesting that inflammation also plays important role in the initiation and aggravation of OA. An early observation by Homandberg and colleagues  suggesteSeveral animal and human clinical studies have shown that chondrocytes, synovium and other surrounding tissues can release pro-inflammatory mediators even in the absence of overt inflammation, and various pathways culminate on aggrecanases and collagenases activation in OA . In OA, Following cartilage damage, the immune system is activated in the joint lining which further triggers synovitis. However, as OA progresses, inflammation can occur surround the damaged joint. This process triggers both innate and adaptive immune systems activation exemplified by the presence of activated macrophages, elevated production of proinflammatory cytokines, activation of complements as well as toll-like receptors (TLRs) which play significant roles in the progression of the disease . PreviouCytokines that have been linked to the pathogenesis of OA include TNF-α, IL-1, IL-2, IL-6, IL-15, IL-17 and IL-21 and several chemokines . Among t2 (PGE2) [IL-1β and TNF-α are also involved in the stimulation of various inflammatory mediators implicated in OA. For instance, chondrocytes treated with TNF-α and IL-1β upregulated the expression of genes encoding inducible nitric oxide synthase (iNOS), COX-2 and microsomal prostaglandin E synthase 1, soluble phospholipase A2, and also stimulated the release of nitric oxide (NO) and prostaglandin E2 (PGE2) . NO and 2 (PGE2) . These c2 (PGE2) . Further2 (PGE2) .Similar to IL-1β and TNFα, IL-6 also regulates the catabolic responses by promoting degeneration of cartilage via the production of MMPs as well as alters the production of type II collagen . IL-6 haThe involvement of TLRs in the pathogenesis of OA has also been reported. A previous study has shown that ECM-derived DAMPS or alarmins act as ligands for TLRs that may lead to the activation of catabolic events and downstream inflammatory responses in articular cartilage . In OA aThe etiology of OA is complex and to date is not fully understood. However, there are few factors that may lead to the occurrence of OA. Age is considered the major independent risk factor of OA. The mechanisms by which age impacts cartilage health are diverse and complex, but they are most likely not just the result of cumulative “wear and tear” over time. As aging progresses, there are changes that occur not only in the cartilage, but also in other joint tissues including synovium, subchondral bone and muscle . Thus, i2 synthesis [Osteoarthritis of the knee is found to be more prevalence in women than in men due to many factors including hormonal influences on cartilage metabolism, gender variation in risk of injury and the differences in the biomechanical activities of the knee . Howeverynthesis  and MMP-ynthesis . In pre-ynthesis . These cynthesis . Sports injury is common in active or young adults and knee injury enhances the risk for OA. As the year progresses 41-51% of participants with a history of knee injury develops radiographic signs of knee OA . Most coThe Osteoarthritis Research Society International (OARSI), the American College of Rheumatology, and the American Academy of Orthopaedic Surgeons (AAOS) have outlined three therapeutic approaches for OA: physical measures, pharmacological therapy and surgery .Physical therapy is related to daily activities. Living a healthier life with a balanced food and physical activities could be an adjunct therapy for OA. Overweight or obese people are at risk should benefit from weight loss as it can reduce the mechanical stress, lessen joint pain, hence reduce OA risks . StudiesPharmacologic treatment in OA is mainly aimed to alleviate pain. According to OARSI and AAOS, acetaminophen is considered as the first-line drug therapy in mild-to-moderate OA . As showNonsteroidal anti-inflammatory drugs (NSAIDs) have long been used to treat moderate-to-severe OA because they provide anti-inflammatory and analgesic properties. NSAIDs function by inhibiting cyclooxygenase-2 (COX-2) and COX-1 enzymes, both of which are involved in prostaglandin synthesis. There are several classes of NSAIDs which include acetylsalicylic acid (aspirin), ibuprofen, naproxen and celecoxib. Eight randomized controlled trials have reported that NSAIDs were superior to acetaminophen in terms of pain alleviation . In a syOpioids such as oxycodone, morphine or atypical opioid tramadol were used for the management of moderate-to-severe pain when NSAIDs and acetaminophen were ineffective or contraindicated . BetweenIntra-articular injections of glucocorticoids and hyaluronic acid is recommended by OARSI as an alternative treatment for knee and hip OA . The intDemands for new drugs for the management of OA are high due to inefficiency and multiple side effects from the current treatments. New drugs are different based upon their therapeutic targets include chondrogenesis inducers, matrix degradation inhibitors, apoptosis inhibitors, osteogenesis inhibitors and anti-inflammatory cytokines . For exa2, resulting in the development of scar-like fibrous tissue or even replacement with bone [Surgery is recommended when both the pharmacology and non-pharmacology treatments for OA fail to reduce pain and restore functions. Total joint replacement is also recommended where the damaged joint is replaced with an artificial one . Total jith bone .The cell-based therapy has been initiated for cartilage repair since 1980s. A technique known as autologous chondrocyte implantation (ACI) was first described by Brittberg and colleagues in 1994 . This teOver the past two decades, three generations of ACI have been developed based on the different types of implantation procedures. In the first generation, the periosteum retrieved from the patient’s tibia were sutured over the defect. The cell suspension was then introduced to the chamber. The disadvantage for the first generation is periosteal delamination. However, in the second generation, it is purely based upon bilayer collagen membrane. This is also sutured over the defect similar to the first generation to form a chamber where the chondrocytes are introduced. More interestingly, in the third generation, cultured chondrocytes are pre-seeded on a 3D-scaffold and then trimmed to fit the size of the defect. Later, it is implanted into the defect area with fibrin glue. Therefore, third generation ACI is called matrix-induced autologous chondrocyte implantation (MACI). The advantages of MACI over conventional ACI is that it reduces the surgical time and the fixation invasion, this in turn gives rise to a long-term cell maintenance.Success stories of ACI have been reported widely. Brittberg  has repoDespite the promising long-term clinical results, there are several limitations to the use of ACI. One of the major limitations is the biological response of the periosteal flap that causes detachment, delamination and late periosteal hypertrophy . However+, CD14−, CD34−, CD45−, HLA-DR−, CD73+, CD90+ and CD105+ [MSCs are multipotent cells that can be differentiated into several types of cells including chondrocytes, adipocytes, osteoblasts and myogenic and neuronal cells ,94,95,96d CD105+ .Isolation of MSCs was first reported by Friedenstein and co-workers through their early works in the 1960s and 1970s ,99. The Unlike chondrocyte cells, MSCs are easy to expand in culture and exhibits chondrogenic potential. The use of MSC-differentiated chondrocytes is a promising strategy for cartilage regeneration. Chondrogenic differentiation from MSCs is well documented from various sources and techniques. Differentiated cells display the important cartilage-specific markers such as collagen type II, aggrecan and sulphated proteoglycans . SimilarGiven their highly chondrogenic potential in in vitro culture, MSC-based therapy is among the promising therapeutic approaches to treat OA. Pre-clinical studies demonstrated encouraging data on therapeutic potential of MSCs in OA animal models. Bone marrow MSCs were implanted onto osteochondral defect which artificially made on 16 rabbits. MSCs-implanted rabbits demonstrated improved histological scores as well as enhanced production of collagen type II in the matrix . Other p® in 60 patients with OA of knee were determined [® was safe, with improved outcome in pain management scores in the lowest dosage (25 million cells). Regardless of its safety and efficacy, intra-articular injection of Stempeucel® especially in the highest dosage (150 million cells) exhibited some adverse effects in the patients, but the adverse effects completely recovered upon symptomatic treatment.In 2002, Wakitani and colleagues transplanted bone marrow MSCs into the articular cartilage defects in knees of 12 patients . Even thtermined . This stAnother source of MSCs that is also utilized for OA treatment is adipose-derived MSCs (ADSCs). As reviewed by Hurley , there wMSC-derived extracellular vesicles (EVs) are a diverse population of heterogeneous membranous vesicles and enriched in many bioactive molecules such as lipids, proteins, mRNAs, transfer RNA (tRNA), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mitochondrial DNA (mtDNA) . These mExosome derived from human embryonic stem cell-induced MSCs (ESC-MSCs) exhibited remarkable cartilage regeneration in osteochondral defects rat which characterized by complete restoration of hyaline cartilage . AnotherBesides having excellent properties for regeneration of tissues, the immunomodulatory properties of MSCs is also one of their superior characteristics. This makes MSCs as a promising cell source to repair the damage of cartilage tissue and at the same time provide immunomodulatory effect to reduce inflammation in OA. MSCs have been extensively studied for their roles in inflammation. MSCs response to inflammation by homing to the damaged tissues, regulating immune and inflammatory responses at the inflamed areas, thus facilitating repair of the damaged tissues . +CD25+regulatory cells [In general, MSCs have the capacity to modulate both innate and adaptive immune responses. MSCs have been reported to modulate cytokine production by the dendritic cell and Th1/Th2 cells , block mry cells . MSCs arry cells ,142. In ry cells . These ary cells . MSCs sery cells .Intra-articular administration of MSCs into arthritic shoulder of the rat model indicated downregulation of ADAMTS5 expression in the joint cartilage, but increased expression of TNF-α stimulated gene/protein 6 (TSG-6) and inhibited the expression of anti-calcitonin gene related peptide (CGRP) indicating suppression of the central sensitization of pain . AnotherMany in vitro and in vivo investigations have shown that MSC-derived EVs have significant anti-inflammatory and regenerative effects in OA models. In an experimental study by Vonk et al., EV isolated from human bone marrow MSC altered the TNF-α-mediated upregulation of COX2 and pro-inflammatory interleukins, i.e., IL-1α, IL-1β, IL-6, IL-8 and IL-17 when co-cultured with TNF-α-stimulated OA chondrocyte . EVs derOA is the most common joint disorder worldwide especially among the elderly. However, OA remains irreversible as there is no effective treatment to cure it. When OA becomes severe, the only option for therapy, other than pain medication, is joint replacement. With current advancement in implants, although joint replacement is an effective therapy for symptomatic end-stage osteoarthritis, outcomes can be poor. The advancements in cell-based therapy offer interesting approaches for the treatment of OA. ACI has been successfully clinically applied in OA patients, however there are a number of issues and concerns surrounding this therapeutic approach as mentioned above. Due to these issues, MSC-based therapy is a promising therapeutic strategy to overcome the difficulty of treating OA. MSCs have chondrogenic properties and exhibit immunomodulatory activity that can help to minimize inflammation in OA. In this review, we described the pre-clinical and clinical studies using different sources of MSC with various approaches. In addition, other approaches using the MSC-based EV in the treatment of OA and their immunomodulatory effects are also described in this review. Despite the fact that MSCs are showing promising results in OA patients, however there are several factors to consider to further improve the treatment and management of the stem cell isolation. The mechanism of action of the stem cell, as well as its chondrogenic potential and immunomodulatory effects in the OA model, must be thoroughly investigated before a new treatment strategy can be implemented. Proper isolation, delivery and management of the stem cells isolated from the patient must be carefully evaluated to avoid immunological rejection and to make sure optimal number of the MSC is obtained. Recently, MSC-based EV offers promising therapeutic potential and immunomodulatory capacity which warrants further investigation. Furthermore, innovative methods using MSC and biomaterial construct for cartilage tissue engineering must be investigated to improve the chondrogenic potential and immunomodulatory properties of the system for OA treatment.


Cardiac point-of-care ultrasound (POCUS) training has been integrated into medical school curricula. However, there is no standardized cardiac POCUS training method for medical students. To address this issue, the American Society of Echocardiography (ASE) proposed a framework for medical student cardiac POCUS training. The objective of this pilot study was to develop a medical student cardiac POCUS curriculum with test scoring systems and test the curriculum feasibility for a future definitive study.Based on the ASE-recommended framework, we developed a cardiac POCUS curriculum consisting of a pre-training online module and hands-on training with a hand-held ultrasound . The curriculum learning effects were assessed with a 10-point maximum skill test and a 40-point maximum knowledge test at pre-, immediate post-, and 8-week post-training. To determine the curriculum feasibility, we planned to recruit 6 pre-clinical medical students. We semi-quantitatively evaluated the curriculum feasibility in terms of recruitment rate, follow-up rate 8 weeks after training, instructional design of the curriculum, the effect size (ES) of the test score improvements, and participant satisfaction. To gather validity evidence of the skill test, interrater and test-retest reliability of 3 blinded raters were assessed.Six pre-clinical medical students participated in the curriculum. The recruitment rate was 100% (6/6 students) and the follow-up rate 8 weeks after training was 100% (6/6). ESs of skill and knowledge test score differences between pre- and immediate post-, and between pre- and 8-week post-training were large. The students reported high satisfaction with the curriculum. Both interrater and test-retest reliability of the skill test were excellent.ClinicalTrials.gov (Identifier: NCT04083924).This pilot study confirmed the curriculum design as feasible with instructional design modifications including the hands-on training group size, content of the cardiac POCUS lecture, hands-on teaching instructions, and hand-held ultrasound usage. Based on the pilot study findings, we plan to conduct the definitive study with the primary outcome of long-term skill retention 8 weeks after initial training. The definitive study has been registered in The online version contains supplementary material available at 10.1186/s40814-021-00910-3. What uncertainties existed regarding the feasibility?2)What are the key feasibility findings?Based on the American Society of Echocardiography (ASE) recommendations, we developed a medical student cardiac point-of-care ultrasound (POCUS) curriculum and assessment tools for the skill and knowledge learning effects of the curriculum to determine the short- and long-term retention. The feasibility of the cardiac POCUS curriculum and the assessment tool validity were uncertain.3)What are the implications of the feasibility findings for the design of the main study?This pilot study confirmed the feasibility of the cardiac POCUS curriculum without recruitment and attrition concerns. The curriculum resulted in large effect sizes of skill and knowledge learning effects. The assessment tools gathered validity evidence from Messick’s framework including content, response process, and internal structure for future use.This formal pilot study provided insight into the medical student cardiac POCUS curriculum and resulted in instructional design changes to enhance the likelihood of success of the definitive study. Based on the pilot study findings, we plan to conduct the definitive study with the primary outcome of long-term skill retention 8 weeks after initial training.https://www.butterflynetwork.com/). The image quality is high enough for cardiac assessment and the costs are touted as affordable to even medical students –[vii].[i] skill test score difference between pre-training and immediate post-training and [ii] the difference between pre-training and 8-week post-training.[iii] knowledge test score difference between pre-training and immediate post-training and [iv] the difference between pre-training and 8-week post-training.We used 5-point Likert scales at immediate post-, and 8-week post-training using Google Forms to measure [v] skill confidence in basic cardiac ultrasound, [vi] knowledge confidence in basic cardiac ultrasound, and [vii] overall curriculum satisfaction.We semi-quantitatively evaluated the feasibility of the curriculum in terms of recruitment rate, follow-up rate 8 weeks after training, instructional design of the hands-on training session, the effect size (ES) of the test score improvements, and participant satisfaction. Feasibility criteria for recruitment rate and follow-up rate 8 weeks after training were 80% or more. We subjectively assessed the feasibility of the instructional design as not feasible, feasible with modifications, or feasible as is . IdentifThe main focus of this study was to develop the cardiac POCUS curriculum with test scoring systems, and then pilot test it on a small sample size of 6 participants to confirm the curriculum feasibility and gain feedback before progressing to a larger scale study. Numerical data of this study could be utilized for sample size estimation for a future definitive study, but the accuracy of the estimation is limited because of the small samples .t-test and presented as mean and SD with 95% confidence interval (CI) and the ES. We interpreted the clinical significance of ES according to Cohen’s effect size guidelines   Mean skill test score difference between pre-training and immediate post-training was 3.56 points  and [ii] the difference between pre-training and 8-week post-training was 2.28 points   and [iv] the difference between pre-training and 8-week post-training was 15.0 points  .The recruitment rate was 100% (6/6 students) and follow-up rate 8 weeks after training was 100% (6/6 students). We identified several instructional design flaws requiring modification of the hands-on training group size, content of the cardiac POCUS lecture, hands-on teaching instructions, and HHU usage. The instructional design modifications are detailed in the discussion section.The 4 skill and knowledge pre-post learning effects  each demonstrated a large ES of 3.05, 0.86, 4.93, and 3.82, respectively. The students reported [vii] overall curriculum satisfaction with mean 5-point Likert scale ratings were greater than 3 points; 4.5 ± 0.5 at post-training and 4.5 ± 0.5 at 8-week post-training (mean ± SD). Based on these feasibility outcomes, we evaluated the overall curriculum’s feasibility as feasible with modifications to implement the future definitive study.This pilot study confirmed the feasibility of the pre-clinical medical student cardiac POCUS curriculum, without recruitment and attrition concerns, with large ES of learning effect outcomes, and favorable student satisfaction with the curriculum. We identified several curriculum design flaws that required modification including the hands-on training group size, content of the cardiac POCUS lecture, the hands-on teaching instructions, and HHU usage.This pilot study trialed hands-on training group sizes of 3 students, 2 students, and 1 student to determine a feasible group size for a definitive study. In the 3-student group size, hands-on training required an excess of 20 min beyond the programmed training time to provide adequate hands-on practice for each student. Moreover, each of the students had 40 min additional waiting time while other students took the pre- and immediate post-training skill tests. We deemed this long waiting time was inappropriate and impractical for volunteer participants. Both the 2-student group and the 1-student group did not raise concerns regarding time management and efficiency. However, during the 2-student group session, we observed the potential for positive or negative peer interactions affecting learning experiences, which could lead to intervention heterogeneity within or between groups, and interfere with consistency and continuity of individualized instructor feedback . A studyTeaching the Teacher (Module E) of the ASE online module recommends a ratio of 4 students to 1 healthy volunteer to 1 instructor as the ideal learning environment (https://aselearninghub.org/). The American Heart Association (AHA) basic life support (BLS) course may provide guidance regarding a practical group size. The BLS course recommends a ratio of 6 students to 2 manikins to 1 instructor which allows instructors to observe the students, provide feedback and guide the students’ acquisition of skills [This study did not intend to determine an ideal or practical group size. The choice of 1-student group size was for feasibility to conduct a future study investigating accurate and unbiased learning effects, not to reveal the optimal group size for learning effectiveness or efficiency. This study also did not seek to identify a realistic or practical group size for curriculum integration into the class size of about 70 students per year in our medical school. We assume it is impractical to implement 1-on-1 sessions for all. At present, there is no evidence regarding an ideal or practical group size for cardiac POCUS training . The Teaf skills . These rBased on student curriculum feedback, we modified the content of the cardiac POCUS lecture for the definitive study. In this pilot study, we included didactic teaching on the cardiac ultrasound beam shape, probe orientation marker, and probe manipulation during the hands-on training, since the pre-training ASE online modules  did not provide content on these topics. We also included a question-and-answer review of anatomical structures seen in the 5 views during the hands-on training to confirm and reinforce student knowledge. Two students gave feedback that these didactic contents would be better provided during the lecture rather than during the hands-on training in order to confirm prerequisite knowledge before hands-on practice and focus on manual skill acquisition during the hands-on training time. In the definitive study, we plan to incorporate the items not involving direct hands-on practice into the cardiac POCUS lecture for effective use of hands-on training time. The modified content of the cardiac POCUS lecture is in Additional File Mean skill test scores for PSAX at 8-week post-training and A4C score at immediate post-training were less than 1.0 point, which means poor or unacceptable for clinical use Table . We planFirst, you start from a clear image of PLAX. Second, you rotate the probe 90 degrees clockwise, and then you usually find the fish mouth appearance of the mitral valve level in the short-axis view. Third, you tilt the probe downward carefully until you see 2 papillary muscles and you should get a clear image of the papillary muscle level of the parasternal short-axis view.During A4C teaching in the study, in order to demonstrate a true longitudinal LV cavity, we instructed students to tilt up a probe adequately after placing the probe on an intercostal space without distorting the image. However, we observed that distorted images after adequate probe tilting was unavoidable for the healthy volunteer A4C image acquisition with the HHU. Therefore, we accepted a mild LV foreshortening and plan to prioritize clear visualization of all 4 chambers in order to provide straightforward instructions to novice students for the definitive study. Our modification of the A4C teaching instruction was supported by ASE POCUS and comprehensive TTE guidelines , 26.The SIVC teaching instruction resulted in excellent quality image acquisition by all students immediately after training Table . HoweverWe experienced automatic ultrasound scan shut-off due to probe temperature increase after continuous scanning during the hands-on training. Once the probe heated up and shut down, we had to suspend scanning for several minutes until the probe cooled down . To manacontent, response process, internal structure, relations to other variables, and consequences. In our study, content evidence refers to the relationship between the test content and what is intended to measure; response process evidence assesses test quality control or the extent to which the test administration and scoring are controlled or standardized to reflects the observed performance; internal structure evidence evaluates the reliability and reproducibility of the test results; relations to other variables evidence evaluates discriminatory ability of the test between experts and novices or the correlation between the test results and known measures of competence; and consequences evidence refers to downstream effects of test results and the decisions that are made [content evidence. Our control measures for obtaining the ultrasound video clips involved asking the students to record when they perceived they had obtained their “best” view, allowed for a second chance to record another view, and had the student choose which of the 2 recordings to submit for assessment. The students controlled the recording, without instructor intervention, therefore adding to the response process evidence of the skill scoring system. Our skill test scoring system demonstrated excellent interrater reliability and test-retest reliability which contributes to the internal structure evidence of this tool. With regard to relations to other variables evidence, we did not assess the test scoring system discriminatory ability between experts and novices for echocardiography because of small novice samples. We planned to gather the evidence in the definitive study with sufficient novice samples. With respect to consequences evidence, we did not gather the evidence because our pilot study did not intend to establish a pass/fail cut-off score or identify the clinical impact of the curriculum. Given the evidence for validity demonstrated by our tools, we determine that these scoring systems can be utilized in the definitive study.We gathered validity evidence based on Messick’s framework recommended by the American Educational Research Association (AERA) . AHA alsare made –42. Our Skill test score results demonstrated that 3 students  retained skill 8 weeks after training and that 3  did not Fig. . We cannClinicalTrials.gov (Identifier: NCT04083924).In this pilot study, we measured 7 learning effect outcomes. We needed to select the most important single primary outcome measure or question in order to establish a primary hypothesis for the definitive study. Our pilot study demonstrated short-term knowledge and skill retention and long-term knowledge retention with a large ES and the 95% CI not including zero, with consistently favorable student confidence and the curriculum satisfaction ratings. However, the skill retention remains unknown due to the small sample size, even though the ES of the skill retention was large. Most previous studies investigating the learning effect of cardiac ultrasound curriculum showed psychomotor or cognitive skill outcomes were improved immediately after an initial training , 43–50. This pilot study confirmed the feasibility of the ASE-recommended medical student cardiac POCUS curriculum, without recruitment and attrition concerns, with large ESs of skill and knowledge learning effect outcomes, and favorable student curriculum satisfaction. The skill and knowledge test scoring systems gathered validity evidence. We modified several curriculum design areas including the hands-on training group size, cardiac POCUS lecture content, the hands-on teaching instructions, and HHU usage. Based on the pilot study findings, we plan to conduct the definitive study with the primary outcome of the long-term skill retention 8 weeks after initial training.Additional file 1. CONSORT 2010 checklist of information to include when reporting a pilot or feasibility trial.Additional file 2. Original content of cardiac POCUS lecture.Additional file 3. Main instruction points for 5 cardiac POCUS views image acquisition.Additional file 4. Skill test scoring system development in detail.Additional file 5. 40-point maximum knowledge test scoring system.Additional file 6. Modified content of cardiac POCUS lecture.


During the coronavirus disease 2019 (COVID-19) pandemic, our aim was to protect staff and patients, therefore, face-to-face clinics were converted to telephone clinics. We retrospectively compared two groups of patients: those seen in traditional clinics pre-COVID-19 and those who had telephone clinics during the pandemic. The mean Ashford Clinic Letter Score (ACLS) for the face-to-face clinic letters was 6.7, and the letters from both groups of telemedicine appointments scored better; the first group scoring 7.1 and the second 7.45. The pandemic allowed us to show that telephone clinics are effective and can be superior to traditional clinics in a specific set of patients. In the past few years, telemedicine/telehealth has become more acceptable and is present in more than 125 countries, even though, in the past, telemedicine has had challenges and some resistance despite great potential -4.As telemedicine was becoming an integral part of our everyday duties at work, we wanted to determine whether telephone clinics are safe, effective, and more efficient compared to face-face clinics regarding making a diagnosis, organising appropriate investigations and making an appropriate treatment plan for the patient using the Ashford Clinic Letter Scoring System (ACLS). This tool has proven to be reliable, reproducible, and concise, which aids in objectively assessing and auditing the quality and efficacy of consultations .In this study, we retrospectively compared two groups of patients: those seen in traditional clinics pre-COVID-19 and those who had telephone clinics during the COVID-19 pandemic. We collected 60 fracture clinic letters from a one-week period prior to the COVID-19 pandemic in March 2020. The clinical correspondence included consultations carried out by the consultant, middle-grade trainees and associate specialists. During this period, the whole service was face-to-face clinics, and this helped reduce selection bias. Cases included a mixture of new trauma referrals and trauma follow-ups, which included operative and non-operative patients.During COVID-19, all clinics were converted to telephone clinics unless a patient required a plaster change or wound check. We then collected 60 fracture clinic letters from telephone consultations from a one-week period at the start of the pandemic in April 2020. Lastly, we collected another 60 fracture clinic letters from telephone consultations from a one-week period one month later in May 2020. We carried out the third set of collection of clinic letters to assess two things: was there a decrease in the number of new patients being referred to the fracture clinic during the pandemic and were clinicians becoming more confident to discharge and make final plans for patients over the phone.All letters were scored using the ACLS guidelines Figures -2. All dThe physical appointment cohort comprised 60 patients; 24 were new patients and 36 follow-up consultations, of which 20 were postoperative patients. In comparison, the first group of telephone consultations only had 11 new patients, and 23 of the patients were postoperative patients. The second group had no new patients and 29 of the patients were postoperative. See Figure There was a clinical working diagnosis in 87% of the face-to-face appointment patients, 90% of the first group telephone appointment patients and 100% of the second group of telemedicine patients Figure . The relFifty-five per cent (55%) of patients seen in the face-to-face clinic already had required investigations available (score 2), 8% had urgent relevant investigations arranged during the same appointment, 33% had investigations required in weeks and 3% in months. In contrast, 80% of patients with telephone appointments had required investigations available, 18% needed follow-up investigations in months and only 3% needed urgent investigations to be arranged immediately.Across the three cohorts, only the face-to-face clinic group had 11.7% of the patients listed for a surgical procedure; the telephone consultation group had no patients listed for surgery. The discharge rate from the face-to-face clinic was about 25%, with 10% of patients sent for further investigations and 50% booked for routine follow-up appointments. In comparison, the initial telephone appointment group had a similar discharge rate to the face-to-face appointment cohort (26.7%) but a significant tendency towards routine follow up (73%). The second telephone appointment group fared better than the other two groups with only 50% routine follow-up (matching face-to-face clinics), and a 45% discharge rate, with the remaining patients sent for further investigations. See Figure The mean ACLS score for the face-to-face clinic letters was 6.7; the letters from both groups of telemedicine appointments scored better, the first group scoring 7.1 and the second 7.45. The combined telemedicine appointment letters scored better than the face-to-face clinic letters (7.275). An independent-samples unpaired t-test was conducted to compare the ACLS mean scores for the face-to-face appointment and telemedicine clinic letters. There was a statistically significant difference in the mean scores for face-to-face and telemedicine letters (p < 0.001) , the question that the clinician is trying to answer during a consultation is ‘What is the diagnosis?’ so that appropriate investigations, treatment and follow-up can be planned . In our It is important to note that there were no new patients in the second group of telephone clinic patients. This could suggest that during the peak of the COVID-19 pandemic, there were fewer presentations of orthopaedic trauma to the Accident and Emergency (A&E) department requiring referral to the clinic, which could have been secondary to the lockdown. We did not investigate how many orthopaedic injuries were seen during this time in the A&E department, which could have helped confirm whether there were fewer presentations. It is equally essential to note that new patients could have been pre-triaged to the face-to-face clinic, however, the volumes of new patients seen in the physical appointment clinics during this period were significantly lower than during pre-COVID times.Most telemedicine patients (80%) already had investigations available on the system from a previous visit, as the majority were follow-up patients. As expected, the face-to-face appointment group endured more emergency (during the same appointment) or urgent (within weeks on follow-up) investigations. This could suggest that face-to-face appointment patients may undergo unnecessary repeat radiographs or further investigations. As our study was limited to purely assessing the ‘quality’ of the consultation using the ACLS, we did not investigate the indications for these additional investigations and whether they were appropriate. On the other hand, having a telemedicine appointment did not impede or delay any relevant clinical investigation, and in most instances (18%), such investigations were only required in a long-term follow-up appointment. These results suggest that patients who do get an initial face-to-face appointment, and/or those that have had an operation can be safely and effectively followed up with subsequent telemedicine appointments once the diagnosis/treatment plan has been established.The discharge rate from the face-to-face appointment was 25% as compared to the discharge rate of 36% across the combined telemedicine group. This discharge rate is similar to an overall discharge rate, which varies from 33%-60% from virtual fracture clinics . AlthougThe mean ACLS scores from the telemedicine appointment letters suggest that telephone consultation for routine fracture clinic and post-operative follow-up of trauma and orthopaedics patients is safe, effective and comparable or even superior to face-to-face clinic appointments. Nevertheless, there are inherent weaknesses when a clinical letter is used as a surrogate for a consultation; the most significant being poor documentation, which can only be mitigated by prospective studies . Our resAs the COVID-19 pandemic continues, we need to continue to adapt and change our practice accordingly. One important way of doing this with clinics is via telemedicine. We have shown that telephone medicine can provide a valuable diagnosis, allow the organisation of relevant investigations and enable the formulation of an appropriate treatment plan for the patient. Furthermore, the Ashford Clinic Letter Scoring System (ACLS) provides an appropriate, concise way of measuring these parameters objectively. With the pandemic continuing and, in some places, worsening, telemedicine will continue to thrive, to probably become the backbone of clinical practice in trauma and orthopaedics in the future, thereby limiting patient-to-clinician contact and protecting both members of staff and patients. Further studies need to be carried out to evaluate the effectiveness of remote consultations in regard to validated outcome measures scoring patient satisfaction. Ultimately, the future of clinical practice should involve patients electing between telephone and physical consultation, as we have demonstrated both can be equally effective.


Here, we conducted 18F-GE180 PET imaging was performed for 11 rats on the last day of the morphine treatment phase and for 10 rats on the response assessment phase of the behavior conditioning procedure. A 18F-GE180 template was established for spatial normalization of each individual image, and the differential 18F-GE180 uptakes between the drug withdrawal (DW) group and the drug addiction (DA) group, the CPA group and the DA group, and the CPA group and the DW group were compared by a voxel-wise two-sample t test using SPM8.Twelve male adult Sprague–Dawley rats were subjected to morphine withdrawal by the administration of naloxone, and six of them were used to model conditioned place aversion (CPA). P < 0.005). Compared with the DA group, the uptake of 18F-GE180 increased mainly in the hippocampus, visual cortex, thalamus and midbrain regions and decreased mainly in the sensory-related cortices after the administration of naloxone in both the DW and CPA groups. Increased 18F-GE180 uptake was only observed in the mesolimbic regions after conditioned aversion compared with the DW group.Both the DW group and the CPA group spent less time in the conditioning cage during the post-test phase compared with the pretest phase, but only the difference in the CPA group was significant  became more severe in the addiction-involved brain regions but remitted in the sensory-related brain regions after the administration of naloxone, and this NI induced by withdrawal was further aggravated after conditioned aversion formation thus may help to consolidate the withdrawal memory. Drug addiction is mainly characterized by the repeated use of addictive drugs. Recently, accumulating evidence has indicated that glial activation, including microglia and astrocytes, and the associated neuroinflammatory signals significantly contribute to drug addiction , 2, whilPlace conditioning is commonly used in cue-elicited drug craving and the treatment of addiction since the rewarding/aversive effects of the drug administered with the environment play a key role in addiction treatment . Steven 11C-PK11195 as the first-generation TSPO PET ligand made TSPO a surrogate marker of NI . Finally, the normalized 18F-GE180 images were arithmetically averaged to create the template  , 24. BripmtatHEP , and theP < 0.05.Statistical analysis was conducted by GraphPad Prism 8. The normality of the distribution of the CPA scores was tested by Shapiro–Wilk normality test and Kolmogorov–Smirnov normality test. The difference of the CPA scores between multi-groups was analyzed by one-way ANOVA following by Bonferroni post hoc analysis, statistical significance was determined as 18F-GE180 uptakes between the DW group, the DA group and the CPA group in pairs using the toolbox spmtatHEP. A significance threshold was set as P < 0.001 with nonmultiple correction, and K > 20 voxels was set as the cluster extent threshold.To detect the change in the activation of glia after morphine withdrawal and CPA training, a voxel-wise two-sample t test was performed by SPM8 with global mean intensity calculation to compare the different 18F-GE180 template was established . The difference in the CPA group between the pretest and posttest was also statistically significant .As shown in Fig. 18F-GE180 uptake in the bilateral visual and cingulate cortices, right hippocampus, thalamus and midbrain as well as decreased 18F-GE180 uptake in the bilateral sensory, insular and piriform cortices, optic chiasm, left hippocampus and pons compared with the DA group . The reduced CPA score also supported this imaging result: after conditioning training, rodents were even less willing to stay in the compartment where they received naloxone injection. Previous studies manifested that the activation of mesolimbic regions causes the release of dopamine substrates which play an important role in reward mechanism and drug reinforcement , 46, and18F-GE180 uptake mainly in the sensory-related cortices  in both the DW and CPA group rats compared with the DA group. There are two possible reasons for this result. One is that naloxone might not only cause NI but also have an antagonistic effect on morphine [We also detected reduced morphine , and premorphine , 53. We morphine ; in addimorphine . FurtherThere are also some limitations in this study. First, since low tolerance of the female rats to morphine, only male rats were included in this study. This might lead to bias of NI on drug addiction/withdrawal. The influence of sex on addiction still needs to be illustrated in further research. Second is that in this animal experiment, we did not measure the affinity of brain microglia to GE180, because there is no significant difference in the affinity of brain microglia in rats. However, when applied in clinical practice, we consider that microglia with different affinities in human brain will have an impact on the uptake of GE180, which should be taken into account.18F-GE180 template to investigate the conversion of NI by detecting changes in glial activation in the brains of naloxone-induced morphine withdrawal rats and CPA model rats. Specifically, after naloxone treatment, NI became more severe in the addiction-involved regions but remitted in the sensory-related regions, and this NI induced by withdrawal was further aggravated after conditioned aversion formation thus may help to consolidate the withdrawal memory. TSPO PET can well monitor the change of NI during morphine withdrawal course.In this rodent study, we set up a


The high disease burden of influenza in elderly and chronically ill adults may be due to the suboptimal effectiveness and mismatch of the conventional trivalent influenza vaccine (TIV). This study evaluated the cost-effectiveness of quadrivalent (QIV), adjuvanted trivalent (ATIV), and high-dose quadrivalent (HD-QIV) vaccines versus TIV used under the current Korean National Immunization Program (NIP) in older adults aged ≥65 years. We also evaluated the cost-effectiveness of programs for at-risk adults aged 19–64 and adults aged 50–64. A one-year static population model was used to compare the costs and outcomes of alternative vaccination programs in each targeted group. Influenza-related parameters were derived from the National Health Insurance System claims database; other inputs were extracted from the published literature. Incremental cost-effectiveness ratios (ICERs) were assessed from a societal perspective. In the base case analysis (older adults aged ≥65 years), HD-QIV was superior, with the lowest cost and highest utility. Compared with TIV, ATIV was cost-effective , and QIV was not cost-effective . The cost-effectiveness of HD-QIV was robust for all parameters except for vaccine cost. The introduction of the influenza NIP was cost-effective or even cost-saving for the remaining targeted gr3oups, regardless of TIV or QIV. Although many consider influenza a mild, self-limiting viral illness, it represents a serious public health problem because of the accompanying pneumonia and high mortality among the at-risk population . FurtherAnnual influenza vaccination is the most effective strategy to reduce the burden of influenza. For this reason, virtually all industrialized and many developing countries recommend annual vaccinations for high-risk populations ,4,5,6. TThe Korean National Immunization Program (NIP) also focuses on the groups for whom influenza vaccination is particularly recommended. They include the elderly, children aged between six months and 18 years, and pregnant women . A majorTo address these problems and establish an evidence-based influenza vaccine strategy, we analyzed the cost-effectiveness of various influenza vaccination strategies in three target groups: (1) older adults aged ≥65 years, (2) extended older adults aged 50–64 years, and (3) at-risk adults aged 19–64 years with chronic medical conditions.This study simulated four scenarios for older adults aged ≥65 years and three each for adults aged 50–64 years and at-risk adults aged 19–64 years, as described below and as shown in -Program 1 (baseline): all older adults received the trivalent influenza vaccine (TIV) according to the current Korean NIP.-Programs 2, 3, and 4: assume the introduction of a quadrivalent influenza vaccine (QIV), adjuvanted trivalent vaccine (ATIV), or high-dose QIV (HD-QIV) to the NIP instead of the TIV, and target a vaccination rate of 85%.-Program 1 (baseline): individuals receiving influenza vaccination with out-of-pocket expenses (TIV or QIV).-Programs 2 and 3: assume the introduction of a TIV and QIV, respectively, into the NIP with a target vaccination rate of 80%.The coverage of the extended program is assumed to reach 80% (adult groups) or 85% (older adults), whereas the baseline program is expected to remain the same as the actual coverage observed in a recent study. In the recent year of the study, the vaccination rate of adults aged 50–64 years was 41.4%, and that of at-risk adults aged 19–64 years was 35.8% ,19. InflA one-year static decision-tree model was developed for this study. The model structure consists of branching for the clinical and economic outcomes of influenza cases associated with TIV, QIV, or other highly immunogenic influenza vaccine strategies, and subsequent branching of the tree reflects seasonal influenza-related complications, hospitalization, and mortality. Those who were infected but did not visit a clinic were excluded from the model considering their low cost burden and more conservative approach. This model allows the conditional probabilities for each chance node to be defined separately for each cohort .Given that the model compared only cohorts over one year, discounting was not applied to either cost or outcomes; only productivity loss due to early death from influenza was discounted at 4.5% in accordance . The effAge-specific Korean population estimates and all-cause mortality for 2018 were obtained from the Korean Statistical Information Service (KOSIS)  [18]. SeA detailed method for estimating disease burden has been previously published . The preUsing the NHIS claims data for the 2017–18 influenza season, per-person medical expenditures were calculated for the following four scenarios: uncomplicated outpatient, complicated outpatient, uncomplicated hospitalization, and complicated hospitalization. Outpatient costs included the cost of office and emergency room visits, including any visits within 14 days of the initial clinic visit, and prescription drug costs at the same time. However, to exclude possible unrelated costs other than those for the treatment of influenza in the outpatient setting, prescription drug costs were excluded for uncomplicated cases; only oseltamivir cost (USD 17.92) was added. Considering that the influenza rapid antigen test is an out-of-pocket expenditure unregistered in the insurance system, influenza rapid antigen test costs (USD 14.98) were added in both outpatient settings. Hospitalization costs comprised costs associated with inpatient hospital stay, outpatient visits, and prescription drugs within 14 days after the influenza diagnosis.Direct nonmedical costs consisted of transportation and nursing costs. The transportation cost per case of hospitalization or outpatient visit  was assigned the value presented in the Korean Health Panel Survey of 2017 . TranspoThe human capital approach was used to estimate indirect costs based on the cost of workdays lost due to illness. Indirect cost was the sum of the cost of workdays lost in visiting clinics or hospitalization, and income lost due to early death. Data from the 2018 survey reports on labor conditions by employment type , and theIt was assumed that older adults (aged 65 years or older) incurred no loss of productivity for the patients themselves, as they are not usually employed.Vaccine costs were assumed to be $8.19 (adult age) or $7.70 (older adults) for TIV, based on the 2018 NIP procurement price at private and public health institutions and the utilization rate of each health institution. Given that the NIP procurement price of the QIV is not yet known, the private market price ratio of the TIV and QIV (1.7 times above) was applied to the NIP procurement price of the TIV to assume that of the QIV. As the ATIV and HD-QIV are not currently available in Korea, an assumed price is also required. Considering the ratio of vaccine price sold in the United States and the price of ATIV in the past in Korea ,28, it wA full description of influenza circulation data has been previously published . Based oThe baseline utility data for healthy and at-risk populations were calculated from a Korean national study, adjusting for the different distributions of age groups used in the model ,24. As dThe vaccine efficacy of TIV against laboratory-confirmed influenza was derived from the Cochrane Database ,31, withThe relative advantage of QIV was imputed, similar to that previously reported . ConsideTo assess the robustness of the results, two sensitivity analyses were performed with major input parameters: a one-way sensitivity analysis to determine which variable had the greatest impact on the cost-effectiveness results and a probabilistic sensitivity analysis (PSA), which assesses the level of uncertainty of the variables in combination within the model. The PSA was performed using a Monte Carlo simulation with 5000 iterations by selecting the input parameter values from a probability distribution . An ICERHerd protection effects were not included in the base case analysis. However, an additional analysis was performed to consider herd effects by calibrating the estimates reported in previous analyses ,39. WithIf the target vaccination rate (80%) was reached in children compared to the current vaccination rate (55%), the relative risk of infection in other age groups was calculated as 0.92–0.9851 using these formulas ,41. GiveFor each vaccination strategy, data on the estimated influenza disease burden, cost, and utility are shown in In the base case analysis, HD-QIV is expected to be a superior strategy compared to all alternatives, with the lowest cost and highest utility. A comparison with the current vaccination rate (TIV 85%) is shown in In the PSA, where the vaccine cost was fixed and all other major parameters were changed, 67.9%, 79.2%, and 92.2% of the simulations fell below the one-GDP per capita WTP threshold for the QIV, ATIV, and HD-QIV strategies, respectively . ConsideA comparison with the current vaccination strategy is shown in However, from the healthcare sector perspective, the introduction of TIV or QIV into the NIP (targeting a coverage rate of up to 80%) is not expected to be cost-effective  .Considering the current trend in South Korea, it is highly likely that QIV will be adopted if NIP is introduced to the expanded adult age group. Thus, a one-way sensitivity analysis was conducted for strategy switching from baseline to QIV (80%), from a societal perspective B. InflueConsidering the herd effect, implementing TIV or QIV into the NIP is still expected to be cost-saving or cost-effective compared to the current state, even under the maximum herd effect .A comparison with the current vaccination strategy is shown in Even from the healthcare sector perspective, the introduction of TIV into the NIP (targeting a coverage rate of up to 80%) is expected to be cost-effective . In comparison, the introduction of a QIV into the NIP is not anticipated to be cost-effective  from this perspective .The results of the one-way sensitivity analysis (in terms of ICERs) from a societal perspective are presented in Even when the herd effect is applied to the maximum, introducing either TIV or QIV into the NIP (targeting a coverage rate of up to 80%) is expected to be cost-saving compared to the current state.This study estimated the disease burden of influenza using the NHIS claims data to assess the clinical and economic impact of various influenza vaccination strategies for three target groups: older adults (≥65 years), adults (50–64 years), and at-risk adults (19–64 years). The results showed that HD-QIV had the most favorable cost-effectiveness profile among all existing commercially available alternatives for older adults, based on currently available data. A universal substitution of TIV with ATIV in the Korean older adult population is also expected to be cost-effective given an ICER of less than $34,767/QALY. Regarding other adult age groups (aged 50–64 years and at-risk adults aged 19–64 years), introducing the influenza vaccine into the NIP is expected to be cost-effective, notwithstanding the vaccine formulation, from a societal perspective. The results of this study are consistent with previous cost-effectiveness analyses of influenza vaccines targeting adults and older adults. Research has shown that influenza immunization programs, especially those adopting QIV, would be cost-beneficial for those with underlying illnesses, even though one of them assumed an influenza vaccine efficacy as low as 32% ,43,44. MHowever, unlike the research conducted so far ,44,46, tThus far, data on the effectiveness of adjuvanted and high-dose vaccines are limited, and the vaccine price is also dependent on estimates. In this study, the efficacy of the high-dose vaccine was determined to be slightly higher than that of the adjuvanted vaccine based on the available literature. The prices of the two vaccine formulations were the same based on overseas sales prices. Therefore, the high-dose vaccine showed a more favorable cost-effective profile than the adjuvanted vaccine. The cost-effectiveness of adjuvanted vaccines should be re-evaluated after comparison with other vaccines, and the Korean price is established. Nevertheless, estimating the acceptable price range of highly immunogenic vaccine formulations compared to the existing ones (TIV or QIV) provides useful information for future vaccine introduction. This study suggests that switching to HD-QIV is cost-effective when the price difference from TIV is less than $12.89 at the currently applied level of vaccine efficacy (relative risk 0.76 compared to TIV). Switching to ATIV is also cost-effective when the price difference from TIV is less than $7.73 at the currently applied level of vaccine efficacy (relative risk 0.8 compared to TIV).1.An “age-based” strategy might be a more efficient option considering the wide range of comorbidities, uncertainty of diagnosis, and implementation issues with influenza vaccine administration within a short period (1–2 months). 2.The cost of the government’s investment in the vaccine program ranged from $242 million (TIV 80%) to $297 million (QIV 80%) for 50–64-year-old adults and from $114 million (TIV 80%) to $140 million (QIV 80%) for at-risk 19–64-year-old adults. Therefore, the total cost to be invested is expected to be lower for the at-risk adult group than for the 50-to-64-year-old group, but implementation issues should be considered.3.From a societal perspective, introducing the influenza vaccine into the NIP (TIV or QIV) appears to be a cost-effective or even cost-saving strategy for both adult age groups. However, after excluding productivity loss, the ICER of introducing QIV into the NIP was $86,463/QALY for 50–64-year-old adults and $53,050/QALY for at-risk 19–64-year-old adults, which is no longer cost-effective for both groups with respect to GDP per capita. Considering that the ICER of at-risk 19–64-year-old adults was lower than that of the comparison group, it might be appropriate to expand the immunization program to the at-risk population first. Of course, it would be difficult to directly determine the dominance between the target groups with an ICER only derived from different analyses; however, it might be meaningful because the analytic results were derived from the same data source and analytic methods. For the expanded adult age group (50–64 years old) and the at-risk adult group, the introduction of the influenza vaccine NIP targeting a vaccination rate of up to 80% is expected to be cost-saving compared to the current state. However, from the healthcare sector perspective, only the introduction of TIV for at-risk 19–64-year-old adults was found to be cost-effective. When expanding NIP within a limited healthcare budget, costs and benefits within the healthcare system might be more important than indirect costs (productivity loss). In addition, several factors must be considered when deciding the priority between the two adult groupsAs expected and evident from the one-way sensitivity analyses, certain key parameters influenced the analysis results. In most analyses, vaccine cost magnified the incremental costs per QALY. As cost is a controllable factor, the findings of this study, which estimate the diverging point of vaccine cost that affects cost-effectiveness, are expected to be useful in policymaking. Vaccine efficacy also appeared to play an important role in the results of each model. Considering that the vaccine efficacy of QIV varied according to the proportion of influenza B and vaccine mismatch, the disease burden of influenza B could also be regarded as a major parameter. In addition, the range of influenza incidence also affects the results; cost-effectiveness results may vary depending on the disease burden of the corresponding season. This study had several limitations. First, there are primary limitations concerning the accuracy of estimating the incidence of influenza from claims data using ICD-10 codes. However, these results are consistent with those obtained in previous studies ,47. The In conclusion, the introduction of the influenza vaccine NIP (TIV or QIV) is expected to be cost-effective in the expanded adult age group (50–64-years old) and the at-risk group (19–64-years old). Moreover, this study indicates that highly immunogenic vaccines for older adults are likely to be favored over the standard non-adjuvanted vaccine, based on currently available data. The relative cost-effectiveness of such formulations (ATIV and HD-QIV) should be re-evaluated after establishing their effectiveness and the Korean price.