The diagnosis of acute human immunodeficiency virus (HIV) infection (AHI) plays a unique role in preventing the spread of HIV and ending the epidemic. Acutely infected individuals are thought to contribute substantially to forward transmissions of HIV; however, diagnosing AHI in resource-limited settings has proven to be a challenge. While fourth generation antigen-antibody combination assays have been successful in high-resource settings, rapid point of care (POC) versions of these assays have yet to demonstrate high sensitivity to detect AHI. Newer RNA/DNA based POC technologies are being validated, but the challenge to understand the additional value of these devices depends on the quality of study evaluations, in particular choice of study designs and case mix of included populations. In this commentary, we aimed to review the quality of studies evaluating a new fourth generation rapid test for detecting AHI, to identify general methodological limitations and biases in diagnostic accuracy studies, and to recommend strategies for avoiding them in future evaluations. The new studies that were evaluated continued to report the same weaknesses and biases that were seen in previous evaluations of fourth generation rapid tests. We recommend that investigators design future studies carefully, keeping in mind how diagnostic performance may be influenced by prevalence, population, patient case mixes, and reference standards. Care must be taken to avoid biases specific to diagnostic accuracy studies . To improve on quality, reporting checklists and guidelines such as Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) and Standards for Reporting Diagnostic accuracy studies (STARD) should be reviewed prior to conducting studies. In light of the 90-90-90 United Nations Joint Program on HIV/AIDS (UNAIDS) targets, identifying the estimated 19 million human immunodeficiency virus (HIV) positive individuals worldwide who are unaware of their HIV status remains a priority . Early dCompared to detecting established HIV (post-seroconversion), there are many challenges unique to the detection of acute HIV infection. Due to the lack of detectable HIV antibodies in the blood during AHI, many diagnostic technologies are rendered ineffective. This is of particular importance for high prevalence resource-limited settings, which rely heavily on rapid tests to detect antibodies to HIV. A recent study in South Africa demonstrated that a substantial number of individuals in the acute and early stage of HIV infection are misdiagnosed as negative upon initial screening with rapid antibody point of care tests , highligEfforts to detect AHI have so far focused on identifying HIV RNA or p24 antigen, both of which appear in the blood prior to HIV antibodies. RNA is detectable even earlier than p24 antigen (5–7 days earlier), thus nucleic acid amplification testing (NAAT) is still the most sensitive method of detecting AHI . HoweverEarly infant diagnosis (EID) faces similar challenges for detecting acute HIV, and a point of care NAAT (Alere™ q HIV 1/2 Detect) has been developed for the purpose of EID . This deFourth generation laboratory assays detect p24 antigen in addition to HIV antibodies. These assays are highly accurate and less expensive than NAAT, but have encountered barriers to being developed in a rapid, point of care format.Detection of p24 antigen is challenging due to is its tendency to form immune complexes with antibodies in the blood, a barrier which has been largely overcome with the use of ultrasensitive heat dissociation techniques; however these techniques are difficult to implement into a rapid, point of care diagnostic test prototype ,13. DiffOur previous work evaluated the overall performance of the Determine HIV Combo rapid test (Alere) in a systematic review and meta-analysis . We founTo follow up on our prior meta-analysis, we aim to describe weaknesses in past evaluations of fourth generation rapid tests, evaluate if improvements have been made in recent studies, and address what is needed to improve future evaluations of rapid diagnostic tests for AHI.We identified numerous weaknesses and biases in study design and conduct, that were recurring across a total of 17 studies evaluating the Determine HIV Combo test published over a period of four years (2010–2014) . Many ofThe most frequent sources of bias in the evaluation of the Determine HIV Combo rapid test, as described in our meta-analysis and QUADAS-2  summary , were riWe have outlined below the various biases and limitations in study design which have specific implications in evaluations for fourth generation combination (antigen/antibody) tests.Spectrum bias has the potential to have a severe impact on accuracy estimates in a study. This bias can occur in case-control studies when patients or blood samples are selected based on HIV status, resulting in a case mix of patients/samples that is not representative of the target population. Patients/samples included in this type of analysis are often at extremes of the disease spectrum , which tends to over-estimate test accuracy. This is critical to keep in mind when evaluating fourth generation combination tests , which can detect markers for HIV at different stages: p24 antigen and HIV antibody.An individual who obtains a positive band for p24 antigen, HIV antibody, or both, is considered HIV positive. An individual who obtains a negative result for both p24 antigen and HIV antibody is considered HIV negative. Calculating an “overall” accuracy estimate for this kind of test, therefore, is actually an average of the performance of the two components in a study population, weighted to the proportion of individuals/samples which are antigen or antibody positive. Investigators must therefore be careful of the case mix of patients they recruit, if the sensitivities of the antigen and antibody components are different.Spectrum bias was observed in the evaluations of Determine HIV Combo rapid test, which has a highly sensitive antibody component but low sensitivity for the antigen component and was evaluated in populations with varying case mixes. Using the evaluations of this test as an example, we noted that cross-sectional studies tended to have a higher proportion of patients in the established/post-seroconversion stage than patients in the acute phase (due to the narrow time frame for detecting AHI), resulting in a higher overall sensitivity. In order to increase the sample size to evaluate the p24 antigen component, many case-control studies specifically sought out p24 antigen positive samples, which naturally overestimates the prevalence of p24 antigen that would be found even in the highest prevalence setting, precipitating spectrum bias. In these cases, the overall performance of the Determine HIV Combo rapid test was more heavily weighted to the antigen component of the test , resulting in a lower overall sensitivity.Spectrum bias can be avoided with careful sampling, to ensure that patients/samples represent the full spectrum of disease that would be expected in a population. When previously stored blood/plasma specimens are included, randomizing the selection of these samples can mitigate this risk. In an observational cross-sectional study, a consecutive or random sampling strategy should be considered.Selecting an appropriate reference standard for evaluating the accuracy of fourth generation tests such as the Determine HIV Combo test is particularly challenging, as the Determine Combo test provides independent results for p24 antigen and HIV antibody. The various assays used as reference standards  for the Determine Combo test were third and fourth generation enzyme immunoassays (EIA), third generation rapid tests, NAAT, Western Blot, and p24 antigen assays. If the accuracy of each test component of the Determine Combo is to be evaluated separately (as was the case), then an appropriate reference standard must be selected which can accurately identify the true presence or absence of p24 antigen or HIV antibody, independently. For example, using a third generation test as the sole reference standard is only suitable when evaluating the performance of the antibody component of a fourth generation test, by nature they cannot detect p24 antigen. This was the case for the evaluation conducted by Bhowan and colleagues, where the reference standard algorithm consisted of two third generation rapid tests in parallel . A fourtFor estimating the overall accuracy, an appropriate reference standard for the Determine HIV Combo test would be a highly accurate fourth generation laboratory assay . Conway and colleagues evaluated the Determine Combo rapid test against a fourth generation laboratory assay, with additional supplementary testing for positive specimens , and samples were classified as acute if they were p24 antigen positive but antibody negative  This allLastly, the choice of statistical methods also plays a role in evaluating test performance. For example, advanced Bayesian statistical methods can help overcome limitations in Frequentist statistical methods typically used for correcting biases due to imperfect reference standards and incorrect verification ,22.Given the biases frequently seen in the studies evaluating the Determine Combo, addressing the question of test suitability for detecting acute HIV becomes complicated. The following questions remain: is the accuracy  more likely to be over or under-estimated in light of these biases, and can they feasibly be avoided in studies of diagnostic accuracy?We ask these questions again in the context of a new fourth generation rapid test for HIV developed by Alere, the Alere HIV Combo test  which isSince our review of the Determine Combo rapid test, four published studies have evaluated the new Alere HIV Combo test ,25,26,27We critiqued three studies (excluding the full text in Japanese for lack of translation) using the validated QUADAS-2 method ,17. LimiRisk of bias in patient selection due to the use of a case-control design was expected in the initial evaluations, as there are distinct advantages to evaluating the performance of a novel test using a case-control study, under “ideal”  environment before undertaking field evaluations. Two out of the three studies failed to mention blinding of reference standard results when interpreting the index test ,27, the The evaluation by Fitzgerald and colleagues restricted their samples to those which were (by reference standard) either HIV negative, p24 antigen positive, or HIV antibody positive, but did not include any samples which were positive for both p24 antigen and HIV antibody by reference standard, putting this evaluation at risk of spectrum bias. This evaluation missed a key group of individuals whose antibodies may not yet be picked up by a second or third generation antibody assay, but could be captured as positive through the p24 antigen component of the Alere HIV Combo test.High quality studies, especially field evaluations, evaluating the Alere HIV Combo (or any similar fourth generation rapid test) in the future will be essential to diagnosing AHI in low and middle income settings. Quality issues can be avoided during the design stage of research studies. To avoid the biases which plagued evaluations of the Determine HIV Combo rapid test, we propose the following recommendations for future evaluations of Alere HIV Combo.The use of a cross-sectional study design is recommended. It is essential to understand how a diagnostic test, particularly rapid and point of care tests, will perform outside of a controlled environment, in field settings. The advantages of using cross-sectional designs compared to case-control studies involve avoiding spectrum bias, obtaining generalizable results, and a more realistic indication of how a diagnostic test is likely to perform once it has been implemented in a health care setting. Cross-sectional studies (even with large sample sizes) may face difficulty recruiting sufficient numbers of patients with acute HIV infection to evaluate the performance of fourth generation rapid tests (such as Determine HIV Combo) during AHI . HoweverIn contrast, there are advantages to using a case-control design when initially investigating a novel test or perfecting a prototype, for achieving an adequately large sample size of p24 positive specimens. The short window period of acute HIV detection (2–4 weeks) makes it challenging to capture a sufficient sample size of acute specimens . It is understandable as to why many researchers turn to a case-control design over a cross-sectional design, particularly in early evaluations where achieving perfection of device prototype becomes the guiding principle for future studies. Regardless of study design, researchers should make efforts to include all types of samples from participants of varying risk profiles, in different stages of HIV infection, not just those which are very clearly positive.Finally, STARD is a reporting checklist for diagnostic accuracy studies ; QUADAS-While we wait for the results of future high quality studies evaluating new tests for acute HIV infection, it is worth mentioning alternative approaches. These approaches are being used to identify acutely infected individuals in resource-limited settings, which may prove useful in the absence of a high performing fourth generation rapid point of care test. One such approach being used in sub-Saharan Africa is targeted screening of individuals, based on signs and symptoms associated with AHI   This appTo conclude, a highly sensitive fourth generation combo rapid test has the potential to improve the detection of acute infections, however overall study quality  needs to be improved in future evaluations. When designing studies to evaluate a novel device, the correct population (case mix) should be targeted to avoid spectrum bias, and investigators should take special care to blind reference standard results when interpreting the index test results. Being mindful of biases and all the key items mentioned above, will not only improve the quality of evaluation, but raise the standard of diagnostic studies for detection of AHI.


In the context of the adult myocardium, which exhibits a limited capacity for 4 Indeed, while the relationship between cell metabolism, substrate utilization and death is profound, the metabolic sensors and regulators that couple mitochondrial substrate utilization for ATP synthesis and cell survival in cardiac cells remain cryptic. In contrast to other cells, cardiac myocytes rely on mitochondrial fatty acid oxidation as the principle fuel source for ATP synthesis. The issue of mitochondrial bioenergetics is of particular importance in the heart as there is a continual demand for high ATP levels by cardiac myocytes for generating contractile force. This is best exemplified by the increased ATP demand by cardiac myocytes during exercise, which require additional cellular energy to sustain the increased cardiac workload. Under normal basal conditions mitochondria produce sufficient ATP to fully meet the energy demands of the cell, but retain the capacity to increase ATP synthesis upon increased energy demand. This ability of mitochondria to increase ATP production commensurate with demand is referred to as ‘spare or reserve' respiratory capacity (RRC). The inability of the cell to sufficiently recruit RRC results in an energy deficit that culminates in cell death.Although the mitochondrion has been identified as a central organelle for integrating substrate utilization and ATP synthesis for normal cellular function, it has also been identified as a critical signaling platform for initiating and executing cell death.2 for driving electron transport chain (ETC) flux and mitochondrial ATP synthesis. The rate limiting step for this process is invariably the conversion of pyruvate to acetyl-CoA by mitochondrial PDH. Indeed, PDH is the gate-keeping step that regulates conversion of pyruvate to acetyl-CoA. In the absence of this critical conversion step pyruvate is metabolized in the cytoplasm to lactate. Notably, PDH is inhibited by pyruvate dehydrogenase kinase (PDK). Hence, elevated levels of PDK inhibit mitochondrial pyruvate flux and mitochondrial TCA-linked respiration by phosphorylating and inhibiting PDH. Though four isoforms of PDK have been identified, PDK2 and PDK4 isoforms are expressed in heart. How PDKs regulate PDH mitochondrial coupled oxidative metabolism and RRC in the heart remain undefined.Despite the vast literature on adaptive mitochondrial programming linking AMP-activated protein kinase (AMPK) and pyruvate dehydrogenase (PDH) to mitochondrial oxidative metabolism in cardiac myocytes, there is a paucity of available information regarding how these metabolic sensors and regulators actually impact mitochondrial oxygen consumption rates (OCR), oxygen-linked ATP production and RRC. Moreover, even less is known of how substrate-metabolic associations link mitochondrial metabolism and respiratory capacity to cell survival during hypoxia or ischemic stress. Under normal aerobic conditions the heart generates ATP by mitochondrial oxidation of fatty acids as the principle fuel source, however, during hypoxia cardiac metabolism shifts from fatty acid to glucose metabolism where glycolysis is the principle source of ATP. Complete glucose oxidation involves the conversion of glucose via glycolysis to pyruvate, which is then fed into to the mitochondrial Krebs cycle  for generating electron donors NADH and FADHet al. address this point in a series of well-designed and carefully executed studies. The authors demonstrate a connection between substrate utilization and mitochondrial respiratory activity. In fact, the seminal finding of the study is the demonstration that cellular substrate can directly influence RRC in a cell- and context-specific manner. For example, neonatal myocytes required both glucose and fatty acids for developing RRC, while human-induced pluripotent stem cell-derived myocytes required only fatty acids for developing RRC that was dampened by addition of glucose. This finding is concordant with metabolic phenotype of the adult heart. This metabolic dependence of RRC was further validated by the demonstration that the metabolic sensor AMPK and metabolic regulators PDH, and the NADH-dependent class III deacetylase Sirtuin-3, positively regulated RRC in cardiac myocytes. This finding is concordant with recent reports demonstrating the ability of RRC to be regulated by these metabolic sensors.6 The data are novel because it demonstrates that RRC can be regulated in substrate-dependent manner in cardiac myocytes. Concordantly, the authors found that hypoxia compromised oxidation of substrates and abolished RRC, with only having incremental effects on basal OCR. Notably, activation of AMPK by AICAR or PDH via the inhibition of PDK4 with dichloroacetic acid (DCA), rescued RRC and OCR.Herein, the manuscript by Pfleger 78 in patients with perioperative lymphopenia 9 or influence the adaptability of human adipocytes to hypoglycemia.10Despite these interesting findings, the underlying source of the mitochondrial RRC in cardiac myocytes remained elusive. In an attempt to address this point, the authors reasoned that RRC may be coupled to a ‘factor' that increases substrate flux coincident with increased metabolic demand on the cell through TCA and ETC flux. In a variety of complementary studies, the authors systematically tested this notion and identified the ‘x-factor' to be succinate dehydrogenase (Sdh). Notably, Sdh is synonymous with complex II of the ETC. Indeed, the exciting feature of this finding is that Sdh is the only enzyme commonly shared between the TCA cycle and ETC thereby providing the ‘missing link' that couples substrate utilization to mitochondrial ETC and respiration. Perhaps most compelling evidence to support Sdh as the elusive factor ‘x' for regulating RRC was the demonstration that RRC was completely abolished by inhibiting the Sdh catalytic activity or by genetic knockdown of the Sdh assembly factor Sdhaf1, which is required for complex II activity. Hence, these findings not only substantiate the importance of complex II in TCA cycle but also importantly provide a functional link between complex II and RRC,13 and support the notion that loss of RRC may underlie disease. Thus, optimizing metabolic substrate availability, by enhancing glucose or fatty acid oxidation by either inhibiting PDKs or activating AMPK, respectively, may prove beneficial in increasing RRC and survival. Alternatively, increasing complex II stability during cellular stress may provide a more direct approach for enhancing or preserving RRC.Collectively, the current data together with earlier work in neuron and fibroblasts demonstrate impaired RRC correlates with increased cell deathAlthough the authors have demonstrated a role for complex II in RRC, the possibility exists that defects in RRC during cellular stress such as hypoxia may directly or indirectly influence RRC. Alternatively, limited availability of TCA reducing intermediates entering the ETC could potentially impair RRC. For example, the depletion of one or more TCA substrate during cellular stress or high metabolic demand during exercise could compromise complex II activity and RRC. As complex II is comprised of multiple subunits, mutations in one or more of these subunits could presumably impair RRC and ATP synthesis. It would therefore be interesting to test whether complex II defects underlie the impaired RRC and ATP deficit commonly seen in human hearts with pre-existing mitochondrial disease. Another area of interest is the relationship between AMPK as a metabolic sensor of cellular stress and complex II activity, and remains unexplored. Notably, as mTOR that drives cellular processes associated with mitochondrial biogenesis and cell growth is regulated by AMPK, it would be important to test whether mitochondrial biogenesis during cardiac hypertrophy in response to systemic metabolic conditions such as diabetes influence RRC and cell survival. Interestingly, Sirt-3 knockdown inhibited AICAR- and DCA-induced RRC, it would be interesting to know whether Sirt-3 influences RRC by deacetylating one or more complex II proteins.Further, given the observed differences in RRC and substrate utilization between neonatal cardiac cells and human iPS cells, it remains untested whether the observed effects on RRC by complex II is universally conserved or a restricted feature of cardiac myocytes. For example, it would be interesting to test whether cancer cells that preferentially generate ATP through aerobic glycolysis would exhibit similar regulation of RRC by complex II during nutrient or hypoxic stress.Nevertheless, under the conditions tested the authors provide new compelling evidence that RRC and cell survival are regulated by mitochondrial complex II activity in a substrate and cell type-dependent manner. The study fundamentally may explain how mitochondrial metabolism linked through complex II activity influences RRC, ATP production and cell survival of cardiac myocytes during hypoxic stress.


In October 2017, the International Olympic Committee hosted an international expert group of physiotherapists and orthopaedic surgeons who specialise in treating and researching paediatric ACL injuries. Representatives from the American Orthopaedic Society for Sports Medicine, European Paediatric Orthopaedic Society, European Society for Sports Traumatology, Knee Surgery & Arthroscopy, International Society of Arthroscopy Knee Surgery and Orthopaedic Sports Medicine, Pediatric Orthopaedic Society of North America and Sociedad Latinoamericana de Artroscopia, Rodilla y Deporte attended. Physiotherapists and orthopaedic surgeons with clinical and research experience in the field, and an ethics expert with substantial experience in the area of sports injuries also participated. Injury management is challenging in the current landscape of clinical uncertainty and limited scientific knowledge. Injury management decisions also occur against the backdrop of the complexity of shared decision-making with children and the potential long-term ramifications of the injury. This consensus statement addresses six fundamental clinical questions regarding the prevention, diagnosis and management of paediatric ACL injuries. The aim of this consensus statement is to provide a comprehensive, evidence-informed summary to support the clinician, and help children with ACL injury and their parents/guardians make the best possible decisions. The number of ACL injuries in children is rising.Progress on these issues can only be made based on long-term follow-up in multicentre collaborations. Achieving progress requires a long-term commitment from those who have children’s interests close at heart. Therefore, in October 2017, the IOC hosted an international expert group of physiotherapists and orthopaedic surgeons who specialise in treating and researching paediatric ACL injuries. Representatives from the following societies attended: American Orthopaedic Society for Sports Medicine (AOSSM), European Paediatric Orthopaedic Society, European Society for Sports Traumatology, Knee Surgery & Arthroscopy (ESSKA), International Society of Arthroscopy Knee Surgery and Orthopaedic Sports Medicine (ISAKOS), Pediatric Orthopaedic Society of North America and Sociedad Latinoamericana de Artroscopia, Rodilla y Deporte (SLARD).Clinicians are charged with the responsibility of providing accurate information and effective treatment to this vulnerable population. Sharing information about the potential consequences of ACL injury and treatment in childhood to long-term knee health should be a central part of the shared decision-making process. Adult patients with ACL injury may develop symptoms and signs of osteoarthritis within 10 years of the index injury.‘Long-term outcomes after ACL injury in childhood, including the development of osteoarthritis, have not been studied.’Box 1Relevant consensus statement topic:Injury preventionRelevant consensus statement topic:Diagnostic tests and imagingRelevant consensus statement topics:High-quality rehabilitationSurgical techniquesThe paediatric ACL graftRelevant consensus statement topics:Skeletal age assessmentThe decision for ACL reconstructionRisks associated with ACL reconstructionManagement of associated injuriesRelevant consensus statement topic:Paediatric patient-reported outcome measuresRelevant consensus statement topic:Ethical considerationsInjury management is challenging in the current landscape of clinical uncertainty and limited scientific knowledge. Injury management decisions also occur against the backdrop of the complexity of shared decision-making with children and the potential long-term ramifications of the injury. This consensus statement addresses six fundamental clinical questions regarding the prevention, diagnosis and management of paediatric ACL injuries . By framA modified Delphi consensus process10.1136/bjsports-2018-099060.supp1Supplementary file 1not important at all to of utmost importance. Consensus was defined as a mean ranking of at least eight points for each statement. After the first voting round, statements reaching consensus were removed, so that only statements that failed to reach consensus in the first voting round went through to the second voting round. The statements that finally reached consensus formed the topics that were discussed at the consensus meeting.A two-round consensus process was conducted, involving 19 content experts. Respondents rated the importance of the 18 predefined statements on an 11-point scale ranging from The IOC convened a consensus meeting of 21 experts in Lausanne, Switzerland in October 2017. The experts were identified by the IOC through the AOSSM, ESSKA, ISAKOS and SLARD member societies, and from physiotherapists and orthopaedic surgeons with clinical and research experience in the field. An ethics expert with substantial experience in the area of sports injuries also participated.how can the clinician prevent ACL injuries in children? Prevention of ACL injury is important because of the potential for serious long-term consequences in those who sustain the injury, and because of the increased risk of reinjury to either knee.This section addresses the fundamental clinical question: Substantial advances have been made in the development and application of ACL injury prevention programs across numerous pivoting sports. There is compelling evidence that ACL injury prevention programs work in skeletally mature patients—they reduce the number of athletes who sustain a primary ACL injury, and reduce the number of new ACL injuries among athletes who return to sport after primary ACL injury.The athlete’s biomechanical movement patterns are a key modifiable risk factor for injury. Injury prevention programs target movement patterns by incorporating strength, plyometrics and sports-specific agility training.‘Injury prevention programs should also be implemented early in the athlete’s developmental process.’Injury prevention programs should also be implemented early in the athlete’s developmental process. This will give the athlete the best opportunity to develop strong and favourable movement strategies. One well-established injury prevention program,Well-designed injury prevention programs have the lowest injury rates and injury time loss.how does the clinician diagnose ACL injury in children? High-quality injury prevention programs are the first-line defence against the potential negative short-term and long-term consequences of ACL injury. However, if injury prevention efforts fail, timely and accurate diagnosis is important, since diagnosis is the starting point for effective management planning and shared decision-making. The clinician combines information from the patient’s history, examination and clinical tests, and imaging to build the clinical picture that will inform diagnosis and treatment. Typically, a thorough history and clinical examination will enable the clinician to make an accurate diagnosis.This section addresses the fundamental clinical question: Haemarthrosis (acute swelling in the knee within 24 hours after a trauma due to intra-articular bleeding) following acute knee injury is an important clue suggesting structural knee injury.Diagnosis can be more challenging than in adults because children may be poor historians, they may have greater physiological joint laxity (be sure to examine both knees) and MRI interpretation is more difficult given developmental variants in children.Due to the immature skeleton, children may sustain different knee injuries  than adults.Consider starting the assessment by ordering plain knee radiographs for all paediatric patients with a haemarthrosis/suspected acute knee injury. This is because tibial eminence fractures and an ACL tear can present with a similar history and physical examination findings. It is also important to rule out other paediatric fractures . Perform an MRI to confirm the diagnosis of ACL injury and evaluate other soft tissue structures.‘No isolated question, test or image can accurately identify an ACL injury, every time.’does not mean that the clinician can always reliably rule the diagnosis in.No isolated question, test or image can accurately identify an ACL injury, every time. The measurement tools available to the clinician are not perfect, but they do yield valuable information in the clinical context. Knowledge of the measurement properties of clinical tools helps the clinician balance the information gained from these tools. The negative predictive values of clinical examination and MRI for ACL tear and meniscal pathology are higher than the positive predictive values . This mewhat are the treatment options for the child with ACL injury? Once the clinician is certain of the injury diagnosis, he or she first needs to know the available treatment options, and discuss these options with the child and the child’s parents/guardian, so a shared decision can be made about how best to manage the knee injury.This section addresses the fundamental clinical question: To restore a stable, well-functioning knee that enables a healthy, active lifestyle across the lifespan.To reduce the impact of existing or the risk of further meniscal or chondral pathology, degenerative joint changes and the need for future surgical intervention.To minimise the risk of growth arrest and femur and tibia deformity.The goals of treatment for the child with ACL injury are:Section 4.There are two treatment options that can help the child with ACL injury (with or without associated knee injuries) achieve these goals: high-quality rehabilitation alone  and ACL reconstruction plus high-quality rehabilitation. In this section, the key components of high-quality rehabilitation for the child with ACL injury, and the options for ACL reconstruction surgical technique are described. Potential treatment decision modifiers are outlined in High-quality rehabilitation is a critical component in the management of ACL injury, and the principles of rehabilitation are the same, irrespective of whether the child has had an ACL reconstruction or has elected for non-surgical treatment. Guidance for paediatric rehabilitation is extrapolated from clinical experience and research in adults, although it is uncertain whether adult principles apply to children.‘Rehabilitation must be performed in close collaboration with the child’s parents/guardians.’‘Children are not small adults.’Dynamic, multijoint neuromuscular control is the primary focus of ACL rehabilitation in children. For the youngest patients , there is less emphasis on the development of muscular strength and hypertrophy. During maturation, and throughout the onset of puberty, rehabilitation strategies that more closely resemble those used with adult patients are appropriate, due to the increase in androgenic hormones.‘Rehabilitation must be thorough, and individualised to the child’s physiological and psychological maturity to achieve successful outcomes.’Rehabilitation must be thorough, and individualised to the child’s physiological and psychological maturity to achieve successful outcomes. Emphasise exercises that facilitate dynamic lower limb alignment and biomechanically sound movement patterns. Although this has been successfully implemented in rehabilitation programs for adolescents and adults, it has not yet been documented as extensively in children. The exercises are gradually progressed through phases II and III of the paediatric ACL rehabilitation protocol  as part 10.1136/bjsports-2018-099060.supp2Supplementary file 2Following surgical treatment, the graft type used for ACL reconstruction, and associated injury or surgery to other ligaments, menisci or articular cartilage, necessitate specific adjustments to the rehabilitation program. Rehabilitation programs should be designed to allow the child to participate in his or her team training sessions to maintain the social benefits of staying within the team. Parents or guardians should be active participants in the daily rehabilitation.Rehabilitation for the child with an ACL injury is organised into four phases , with anThe framework for progression through functional milestones is similar for ACL reconstruction and non-surgical treatment. However, there are different expectations for progression and time to return to full participation in sport. For all patients, rehabilitation progression must be guided by clinical and functional milestones , and retData from international registries suggest that young athletes are at high risk for a second ACL injury following an ACL reconstruction,‘Consider advising the child athlete not to return to pivoting sport until at least 12 months following ACL reconstruction.’Box 2PrehabilitationFull active extension and at least 120 degrees active knee flexionLittle to no effusionAbility to hold terminal knee extension during single leg standing For adolescents: 90% limb symmetry on muscle strength testsPhase I to phase IIFull active knee extension and 120 degrees active knee flexionLittle to no effusionAbility to hold terminal knee extension during single leg standingPhase II to phase IIIFull knee range of motion80% limb symmetry on single-leg hop tests, with adequate landing strategiesAbility to jog for 10 min with good form and no subsequent effusionFor adolescents: 80% limb symmetry on muscle strength testsPhase III to phase IV: sport participation (return to sport criteria), and continued injury preventionSingle-leg hop tests: >90% of the contralateral limb Performed gradual increase in sport-specific training without pain and effusionConfident in knee functionKnowledge of high injury-risk knee positioning, and ability to maintain low-risk knee positioning in advanced sport-specific actionsMentally ready to return to sportFor adolescents: 90% limb symmetry on muscle strength testsMuscle strength testing should be performed using isokinetic dynamometry or handheld dynamometry/one repetition maximum. The type of test and experience of the tester are highly likely to influence the results. If using handheld dynamometry/one repetition maximum, consider increasing the limb symmetry criterion cut-off by 10% . Clinicians who do not have access to appropriate strength assessment equipment should consider referring the patient elsewhere for strength evaluation.Consider a home-based program, with emphasis on playful exercises and variation  to discoSingle-leg hop tests and isokinetic strength tests have larger measurement errors in the prepubescent population, so use these tests with caution.Focus on evaluating the quality of movements during single-leg hop testing, instead of the leg symmetry index measures.Tests and criteria to assess movement quality are yet to be validated, so the responsible clinician needs to have skills and experience in this area.Return to sport criteria were designed and scientifically tested in the skeletally mature patient and are recommended for the child who is close to maturity.Children who are close to skeletal maturity may follow rehabilitationMany clinicians involved in non-surgical treatment of skeletally immature children recommend the child wear a protective brace during strenuous physical activities.The general principles of ACL reconstruction in adults also apply to the paediatric patient: use a well-positioned (soft tissue) autograft of adequate size, with adequate fixation to allow functional rehabilitation. Physeal damage should be minimised to avoid growth disturbance. Bone plugs and fixation devices should not cross the physis.The child has repairable associated injuries that require surgery .The child has recurrent, symptomatic knee giving way after completing high-quality rehabilitation.The child experiences unacceptable participation restrictions .There are three indications for paediatric ACL reconstruction:There are three possible techniques for paediatric ACL reconstruction.The transphyseal technique in the child is similar to the technique the surgeon would use for ACL reconstruction in adults. Single bundle transphyseal ACL reconstruction with a quadrupled hamstring graft is the most common .46–51 ThPhyseal-sparing techniques avoid physeal damage in patients with markedly open physes. The techniques include an over-the-top technique with a strip of the iliotibial band ,53 and atransphyseal tibial tunnel with a physeal-sparing technique on the femoral side.The partial transphyseal technique  combinesDrill hole trajectory and location influence the degree of risk to the physes . KnowledDrilling at the periphery of the physis and the perichondral ring increases the risk of growth disturbance. Drill holes may be placed in an all-epiphyseal manner to allow for drilling at the native ACL footprint, while avoiding the physis. Precise tunnel placement is required when performing this technique to avoid damage to the undulating distal femoral physis.Bone tunnel drill holes should be as vertical as possible (while still maintaining anatomic graft position) and as central as possible. This is especially important when drilling through the anteromedial portal. Drilling an oblique tunnel rather than a more vertical tunnel increases the amount of physis removed and increases the risk for growth disturbance.Do not cross the epiphysis with hardware, implants or bone blocks. Fill bone tunnels with soft tissue, rather than leaving the tunnels open.Box 3Advantage: minimises physeal volume affectedDisadvantage: less than ideal coverage of ACL footprintAdvantage: anatomical graft position covering the ACL footprintDisadvantage: greater volume of physis negatively affectedAdvantage: appropriate placement at ACL footprint; no drilling through the physisDisadvantage: requires precise tunnel placement to reduce the risk for physeal damageshould not be harvested in paediatric patients with open physes to avoid damage to the tibial tubercle apophysis. Allografts are not indicated in paediatric patients in most cases, since  the use of allografts in paediatric ACL reconstruction has poor clinical outcomes.Only soft tissue grafts  should be used for ACL reconstruction in paediatric patients with open physes. The quadrupled hamstring graft is most common.‘The use of allografts in paediatric ACL reconstruction has poor clinical outcomes.’A novel technique involving the use of living-donor hamstring tendon allograft has been reportedExtracortical fixation of soft tissue grafts may be performed with a cortical button, suture, post or staple. Aperture fixation may be performed with interference screws, provided the screws do not cross the physis.Data regarding ACL graft incorporation in children are scarce. Paediatric soft tissues have a greater biological growth potential compared with adults,increase diameter as the child grows, but may increase in length.The ACL graft must adapt as the child grows. The graft may increase in length as the bone grows, and the bone tunnels may reduce in relative size.With longitudinal bone growth after transphyseal ACL reconstruction, the graft may become more vertically oriented. This observation might be explained by the movement of the femoral fixation site with physeal growth or because the tibial tunnel aperture becomes relatively more posterior due to greater anterior growth of the proximal tibia. Other changes occurring as the child grows are secondary intercondylar notch narrowing, distal migration of the tibial and/or proximal migration of the femoral extracortical fixations and verticalisation of the Blumensaat line.what are the most important considerations when making treatment decisions? The key issues addressed relate to assessment of skeletal maturity, the decision for surgery or not, management of injuries to other knee structures and potential adverse events following treatment. These issues may alter the ACL injury management decision, depending on the decision-making team’s ) risk tolerance.This section addresses the fundamental clinical question: Protecting the physis and perichondral ring from damage during ACL reconstruction is an important considerationcan result in premature closure.Assessing and documenting the child’s skeletal age, in addition to his or her chronological age, is necessary to individualising treatment of ACL injuries. The main goal with respect to skeletal age assessment is to define remaining knee growth. ‘Estimating skeletal age and remaining growth are key considerations for treatment decision-making.’Box 4Understand the difference between skeletal age and chronological age.Use imaging of the knee to determine if the femoral and tibial physes, and the tibial tubercle apophysis are open. If the growth areas are closed, then, independent of chronological age, the child can be treated as an adult.None of the specific methods for skeletal age determination in isolation is sufficient to accurately determine skeletal age.Use a multifaceted clinical approach to determine skeletal age that includes whether or not the child has had an adolescent growth spurt, the relative heights of the child’s parents and Tanner staging.The most common method of skeletal age assessment is via posterior-anterior left hand and wrist X-ray. This can be compared with a skeletal atlas . The clinician might also consider long leg radiographs (hips to ankles) after injury to establish a baseline for assessing the potential development of angular deformity and leg length discrepancy. Assessing skeletal age is also relevant in research and may be beneficial for medicolegal reasons. If overgrowth, growth arrest or deformity occurs, presurgical documentation of skeletal age may be important .Box 4FivChildren who have repairable additional injuries at ACL injury diagnosis  should be treated with early ACL reconstruction and meniscal repair.A well-performed ACL reconstruction and preservation of the meniscus can restore knee stability.Children who undergo ACL reconstruction after failed non-surgical management may have a higher number of meniscal and chondral injuries at the time of ACL reconstruction compared with those who undergo early ACL reconstruction.‘Non-surgical treatment is a viable and safe option in skeletally immature patients who do not have associated injuries or major instability problems.’Section 3 for the key principles of high-quality rehabilitation). Non-surgical treatment can be a permanent treatment option for those who do not develop functional instability, or a short-term option to delay ACL reconstruction until the child has reached skeletal maturity. Abandoning non-surgical treatment in favour of ACL reconstruction is an option if the child has recurrent instability problems despite completing high-quality rehabilitation, or if the child has a secondary intra-articular injury. Therefore, clinicians must work together to closely and frequently monitor the child with repeated MRI and clinical examination as appropriate, being alert to instability episodes and secondary injuries that require prompt assessment and treatment.Non-surgical treatment is a viable and safe treatment option in skeletally immature patients who do not have associated injuries or major instability problems.Irrespective of the technique, surgical treatment of the ACL has inherent risks. Different ACL reconstruction techniques have different considerations to help avoid risk to the physes, articular surface and soft tissue structures of the knee. Here, we describe five key risks associated with surgical treatment for ACL injury of which clinicians, patients and their parents/guardians must be aware.Growth disturbances are a rare (approximately 2%)increase the risk of rotational deformity and decrease the risk of angular deformity.Highly tensioned soft tissue grafts placed across femoral physes have been associated with limb length discrepancy and angular deformity.Most patients with ACL rupture requiring surgical treatment are approaching skeletal maturity, and do not have substantial growth remaining. This means that angular deformities and limb length discrepancies are likely of relatively low clinical significance. Therefore, it may be reasonable to perform transphyseal procedures when the child has minimal growth remaining.Routine clinical and radiological follow-up within the first 12 postoperative months can help the surgeon detect early clinical and radiographic evidence of leg length discrepancy, angular deformity or and physeal injury. For the child with markedly open physes, appropriate follow-up evaluation of leg length discrepancy might include annual clinical assessment and knee radiographs with long-leg alignment views until skeletal maturity and physeal closure. Height should be monitored, and if growth exceeds 6 cm in 6 months, or if clinical findings warrant, the annual assessment should be brought forward.Localised physis injury resulting in a bone bridge leading to growth arrest and possible malalignment (type A);Overgrowth process potentially caused by hypervascularisation (type B);Undergrowth process arising from a graft traversing a physis under tension during growth and leading to a tethering effect (type C).Growth disturbances can occur in several different forms . The growho returned to pivoting sports after ACL reconstruction can be expected to sustain a new ACL injury .Young age, returning to pivoting sport and receiving an allograft are important predictors of new ACL injury after index ACL reconstruction.Section 3. The ACL graft is also affected by the status of the other ligaments, menisci, cartilage surfaces, limb alignment, rotation and the dynamic muscle control of these structures—all factors that must be considered during treatment decision-making.High rates of reinjury among young people with ACL reconstruction are concerning, although data regarding reinjuries among children with ACL reconstruction are sparse in comparison to data from skeletally mature patients. The best available evidence suggests a graft rupture rate in children and adolescents (age range 6–19 years) of 13%, and a contralateral ACL injury rate of 14%.Meniscectomy is associated with an increased risk for osteoarthritis.Knee stiffness may be due to the degree of injury to the ACL, disruption of the joint capsule and injury to structures other than the ACL. Knee stiffness may also be related to surgical interventions or inadequate rehabilitation. Knee stiffness is rare in children aged 13 years and younger, and less common in males and in those having surgery with an iliotibial band or hamstring autograft.Data related to infection risks for paediatric patients are extrapolated from literature that combines paediatric and adult patients. Infection rates in adult patients are generally low for ACL reconstruction. The rate of deep infections after ACL reconstruction with autograft is 0.19%.Here we address the key issues for managing cartilage and meniscal injuries in combination with ACL rupture, and the multiligament-injured knee.The degree of vascular penetration of the menisci declines with age, with between 10% and 30% of the menisci receiving vascular inflow in adults.It is important to evaluate the MRI characteristics of the paediatric menisci to rule out meniscal injuries. In cases where the diagnosis is difficult, a diagnostic arthroscopy may be performed to clarify the diagnosis and ascertain the state of the meniscus. The clinician should also assess for a posterior medial meniscocapsular tear (ramp lesion).‘The clinician should also assess for a posterior medial meniscocapsular tear (ramp lesion).’Ramp lesions may be present in one in six adult patients with ACL injury, and the prevalence of ramp lesions in children with an ACL injury is similar to adults.‘Meniscal repair should be performed whenever possible’Meniscal repair should be performed whenever possible in the paediatric patient because of the deleterious effects of meniscectomy and the positive outcomes of meniscal repair .Articular cartilage injuries in combination with ACL injury are less common than meniscal tears.There is limited research on multiligament knee injuries and treatment in paediatric patients, and these injuries are less common in children than in adults.Use fluoroscopy prior to placing suture anchors for a repair, or for tunnel reaming for a concurrent ligament reconstruction, to evaluate tunnel position in relation to the physes.Non-surgical treatment may be appropriate for partial posterior cruciate ligament (PCL) tears or non-displaced avulsion injuries. PCL reconstruction is a relatively safe and viable treatment option in patients with multiligament injuries.Perform a reduction by manipulating the tibia relative to the femur. Avoid forceful hypertension or rotation, to minimise the risk for damage to cartilaginous and/or neurovascular structures. Following reduction, a dynamic knee brace can be applied (for at least 12 weeks) to prevent further intra-articular damage and to help hold the knee reducedhow does the clinician measure outcomes that are relevant to the child with an ACL injury? Assessing patient-reported outcome measures (PROMs) provides insights into aspects of the patient’s function that cannot be evaluated with clinical tests or imaging.This section addresses the fundamental clinical question: Valid outcome instruments must have appropriate measurement properties, including reliability, validity  and responsiveness. Instruments that were developed for adults may not be valid for children and adolescents. Paediatric patients have different levels of comprehension (this age group includes a spectrum of comprehension abilities from younger children to older adolescents) and interpretation of instruments. Most importantly, paediatric patients may value different outcomes when evaluating their knee function, and instruments must reflect the issues that are important to children and adolescents.Paediatric PROMs should be either developed or specifically validated in this population. The process of validation should include an assessment of comprehensibility, reliability, validity and responsiveness. Child-reported outcome assessment is typically valid in older children and adolescents (aged >10 years).Paediatric PROMs  must be Use a generic measure of health-related quality of life;Use either the Pedi-IKDC or KOOS-Child to assess self-reported knee function;Use the Pediatric Functional Activity Brief Scale to assess self-reported activity level.Recommendations for using PROMs in clinical practice with paediatric patients:In research, it may be appropriate to include other PROMs depending on the research question. Researchers need to make decisions about the most appropriate outcome(s) when planning their study.what are the clinician’s role and responsibilities? Treatment decisions that involve children are among the most difficult decisions the clinician faces, especially when scientific knowledge is limited. Striking a balance between ethical principles can be especially challenging when there is a conflict of opinion. In this section, we outline the relevant ethical considerations for the clinician who treats children with ACL injuries.This section addresses the fundamental clinical question: It is impossible to provide specific ethical guidance that applies to all sporting injuries in adolescents and children, given the varying individual circumstances. However, it is incontrovertible that it is in the best interests of all children not to have knee and associated injuries. Therefore, injury prevention programs are fundamental to the best interests of the child. Clinicians have an obligation to support policies and practices that encourage coaches, teams/clubs and (inter)national federations to prioritise injury prevention. All parties should be committed to protecting the long-term welfare of the growing child. Nevertheless, there may be exceptional cases where parents/guardians may, with the approval of their child, rationally prioritise short-term goals. One example could be that, despite inherent risks for reinjury, an early return to sport might be a high priority for a child who has exceptional talent in a given sport.Protecting the integrity of the knee should be the clinician’s primary focus. Decisions regarding how to protect the integrity of the child’s knee must be shared between the child, parent/guardian (surrogate decision maker) and clinician.Children are a vulnerable population.The clinician and/or parent(s) are obliged to serve the interests of the child above all other interests.There should be consensus between all parties when arriving at a decision. This consensus should be based on realistic assessments of risks and benefits and a proper consideration of the goals of the child and parent. The clinician’s responsibility is to guide this discussion with accurate information from the best quality research. There are several ethical standards that can help the clinician, child and parent(s) navigate the decision-making process, and arrive at ethically justified treatment decisions.Box 5Best interestsshould not acquiesce to parent-led decision, so that the child is not harmed.Harm principleParental discretionCosts/benefitsNot unreasonableReasonable choiceSome paediatric ethical standards are not identical—some aim at higher thresholds, while others accept a lower threshold of justification. There are six standards that can be helpful in different clinical scenarios in paediatric ACL injury .Box 5SixThe clinician has an important role in treatment decision-making, because he or she typically has superior knowledge of treatment options, risk and benefits than children and parents. To best guide the child and his or her parent(s), the clinician must have a clear idea of the range of interventions that are (1) optimal, (2) acceptable and (3) not desirable, and be able to justify this with reference to the best quality research and clinical experience. In many healthcare settings, parent(s) take responsibility for the ACL treatment decision, commensurate with the child’s assent. Where there is a lack of consensus in the decision-making process , the clinician may also consider whether he or she can defend a treatment recommendation based on one of the six ethical standards.Management of paediatric ACL injuries is strongly debated. Reflecting some of the concern and controversy is a high ratio of clinical commentaries and narrative reviews to original articles on this topic. The problem for the clinician is that there is scarce high-quality evidence that he or she can look to, to help him or her best manage paediatric ACL injuries. The scientific literature is inconsistent and limited by inferior methods that carry a high risk of bias.Most clinical studies on paediatric ACL injury are of cross-sectional or retrospective design, the study populations are often at high risk of selection bias and include small samples. This means there is a high risk that existing research does not reflect the typical paediatric patient with an ACL injury.Many studies do not provide adequate descriptions of the treatments that the patients have received, and patient adherence has not been reported. A meaningful interpretation of study outcomes is only possible with a detailed description of the surgical technique, rehabilitation, brace usage, return to sport clearance and recommendations of activity modification.Many studies fail to assess the skeletal age of included participants, and few report the remaining growth of participants. Chronological age alone is an unreliable indicator of skeletal maturity. Because of this, it is difficult to know to which skeletal age group these research results apply.Patients aged up to 18 years are often included in paediatric studies. This is a problem because it is likely that the patient population is a mix of skeletally mature and immature patients. Therefore, the literature may be biased towards the older patients. Having mixed populations also complicates pooling or comparing results from skeletally immature patients across studies.Knowledge of preinjury and post-treatment activity level gives important insight into a key risk factor for injury. The greater exposure a child has to potentially injurious situations , the greater the chance of (re)injury. Activity level is a key confounding factor that is rarely accounted for in statistical analyses. This means there is a risk that estimates of secondary injury incidence may be overestimated or underestimated in comparisons between studies or patient-groups.There are five key issues that must be addressed by future studies:Prospective injury surveillance studies to identify injury mechanisms and modifiable risk factors for ACL injury, combined injuries and knee reinjuries.Prospective research on outcomes after surgical and non-surgical treatment. Long-term follow-up (beyond 10 years) is essential to answer key questions of how an ACL injury in childhood impacts physical activity, future knee-health and quality of life.Research on the efficacy of different surgical techniques and characteristics , and high-quality rehabilitation programmes, knee brace usage and activity modification after injury and surgery.Multicentre and registry studies should be prioritised. Because of smaller numbers of ACL injuries in paediatric patients than in skeletally mature patients, specialist treatment centres, expert clinicians and researchers must prioritise collaboration.There are four research priority areas to improve prevention and outcomes of paediatric ACL injury:An excellent clinician-scientist and a keen coworker in this project, Allen F Anderson, MD, died in a farming accident on Sunday, 12 November 2017. This tragedy occurred shortly after he had been an active participant in this IOC consensus meeting on the topic of his life-long clinical and research passion, paediatric ACL injuries.Born on 16 November 1949, Dr Anderson was a graduate of the University of Tennessee College of Medicine. He completed a residency in orthopaedics at Vanderbilt University and was board-certified by the American Board of Orthopaedic Surgery in general orthopaedics, with a certificate of added qualification for Sports Medicine.Dr Anderson was a sports medicine specialist with an interest in knee injury and ligament reconstruction, and with special interest in children’s injuries. He published more than 100 peer-reviewed journal articles and 26 book chapters, and received a patent for the invention of a paediatric ACL reconstruction system. Among numerous awards, three standouts were: being recognised as one of America’s Top Physicians 2004–2012 from Consumer’s Research Council, being elected to Best Doctors in America by his peers 2007–2008 and being Nashville Business Journal Top Doctor 2016–2017.The Orthopedic Journal of Sports Medicine and The American Journal of Sports Medicine.Dr Anderson had many prestigious positions through his life. He served as President of the American Orthopaedic Society for Sports Medicine from 2015 to 2016, and as an Associate Editor of Above all, he was a true friend and colleague whom you could go to with problems and challenges, not the least among our youngest patients. Allen will be greatly missed by us all.


A current approach to obtain bioengineered lungs as a future alternative for transplantation is based on seeding stem cells on decellularized lung scaffolds. A fundamental question to be solved in this approach is how to drive stem cell differentiation onto the different lung cell phenotypes. Whereas the use of soluble factors as agents to modulate the fate of stem cells was established from an early stage of the research with this type of cells, it took longer to recognize that the physical microenvironment locally sensed by stem cells  also contributes to their differentiation. The potential role played by physical stimuli would be particularly relevant in lung bioengineering since cells within the organ are physiologically subjected to two main stimuli required to facilitate efficient gas exchange: air ventilation and blood perfusion across the organ. The present review focuses on describing how the cell mechanical microenvironment can modulate stem cell differentiation and how these stimuli could be incorporated into lung bioreactors for optimizing organ bioengineering. Significant respiratory diseases such as chronic pulmonary obstruction, emphysema, idiopathic pulmonary fibrosis, primary pulmonary arterial hypertension, interstitial lung disease, cystic fibrosis and α-1-antitrypsin deficiency result in irreversible structural lung damage, with lung transplantation as the only therapeutic indication when the disease reaches an advanced progression . Hence, This short review aims to address the topic of lung bioengineering, specifically focusing on the potential differentiation effects of the mechanical stimuli sensed by stem cells seeded in decellularized organ scaffolds. The first section briefly refers to acellular lung scaffolds as the currently preferred platform for organ bioengineering. The second section is succinctly devoted to mention that stem/progenitor cells are the ones used to repopulate the acellular lung scaffolds for lung regeneration. Detailed information on stem cells for this application can be found in a very recent review published by this journal . The thiSome potential approaches for engineering functional tissue elements are based on constructs at the microscale level, for instance as described in lung-on-a-chip applications mimicking the alveolar-capillary membrane , or on sCompared to other organs, the structure of the lung is particularly complex, and hence lung decellularization is a complex process. Indeed, the lung is structured as an asymmetrically branching airway tree starting at the trachea and ending at ~300 millions of alveoli. Moreover, there is a whole pulmonary vascular circuit providing blood perfusion to each individual alveolus. These two fluid circuits (for air and blood) are anatomically and functionally matched to facilitate gas exchange through the alveolar-capillary membrane. In addition to its structural complexity, the lung compartments contain up to 60 different types of cells . DespiteIn the last few years, some studies have provided proof of concept for lung bioengineering. Price et al.  reportedUsing undifferentiated cells for seeding a lung extracellular matrix could be a more practical strategy for lung bioengineering given the possibility of expanding these cells and their capacity to differentiate into different phenotypes. However, this approach requires that stem cells differentiate into the required lung phenotypes at each specific site within the organ structure.Interestingly, Cortiella et al.  reportedIn a study carried out on bioengineered rat lung lobes seeded with human adipose tissue-derived MSC (hAT-MSC) and human bone marrow-derived MSC (hBM-MSC) the authors observed that both cell types could adhere to the decellularized matrix . After cOther studies performed in rodent  and humaTherefore, the research experience currently available using undifferentiated cells for lung bioengineering indicates that the decellularized organ scaffold would play a fundamental role in modulating important stem cell processes of homing and differentiation, and specially in modulating the secretion of required ECM components which would contribute to tissue reconstruction .Stem cells, given their potential to expand in vitro and differentiate onto multiple lineages, are an important target in studying organ regeneration. It is currently well recognized that stem cells multi-potency is modulated by signals of the surrounding local microenvironment, including biophysical and biochemical cues presented by the matrix and soluble mediators secreted by neighbor cells or coming from systemic pathways , 21. In In the case of lung cells Fig. , the mosper se. For instance, soft matrices that mimic brain stiffness are neurogenic, while stiffer matrices mimicking muscle are myogenic and more rigid matrices that mimic bone are osteogenic [Substrate physical properties such as stiffness, 3-D structure and viscoelasticity modulate the differentiation of stem cells. It is well accepted that some aspects of the dynamic 3-D cell environment play an important role in unlocking the full potential of stem cells associated with their renewal, differentiation, and assembly which is observed in native tissues –29. Cellteogenic . Moreoveteogenic . Forces teogenic , homogenteogenic . Additioteogenic  and 3-D teogenic  for lungteogenic –39.Cyclic stretch -which can be applied at different frequencies to mimic respiratory rates –42- is aShear stress is of particular interest in cells of the blood vessels. It is known that this mechanical stimulus modulates endothelial function and, potentially, endothelial specification of stem cells , 52. WanThe existence of an air/liquid interface is another factor that can considerably affect stem cell fate into lung phenotypes , 59. Van2), it is well documented that, in vivo, both embryonic and adult tissues, and specifically the different lung compartments, are subjected to oxygen partial pressures far below the ones corresponding to room air [The role played by oxygen partial pressure in stem cell differentiation has been scarcely examined and requires more detailed work –66. Inderoom air . Given troom air , cardiomroom air , chondroroom air , and, moroom air . More sproom air  and muriroom air  MSC haveroom air . This poroom air .Therefore, the existing experimental evidence above mentioned gives support to the concept that the physical microenvironment significantly contributes to modulation of the differentiation of both embryonic and adult stem cells. However, specific data concerning stem cell differentiation towards lung phenotypes by physical stimuli are scarce. In an interesting study, decellularized rat lungs were recellularized with human iPSCs-derived endothelial cells and iPSCs-derived epithelial progenitor cells via pulmonary artery and trachea, respectively. The whole lung was then cultured for 12–15 days under vascular perfusion and the results showed mature lungs phenotypes expressed by Nkx2.1, CD31, T1α/podoplanin and CC10 markers . In anotIt is of note that most reported studies focus on the effect of an individual physical stimulus on stem cell fate. However, cells differentiating in vivo are subjected to a variety of simultaneous stimuli: a myriad of physiological soluble factors and specific molecular composition of the extracellular matrix in addition to different general physical factors such as 3-D architecture and substrate stiffness. In the case of cell lungs, additional mechanical stimuli are active: cyclic stretch, shear stress, air-liquid interface or oxygen gradients. Although there is not much information on the combined effects of these many stimuli on stem cell differentiation, it seems reasonable to expect different potential outcomes: from counterbalancing the effect of each other to additive or synergistic responses. In fact, the relatively scarce information available suggests that the result would depend on what specific mechanical stimuli are combined and on their relative intensity –81. OwinIt is worth noting that a considerable number of studies focusing on how mechanical stimuli modulate the fate of stem cells have been carried out on MSC, probably because, as compared with other stem cells, they are easily obtained from patients even for autologous applications. While these works are of great interest to improve our understanding of the mechanisms involved in stem cell mechanotransduction, their impact on the specific field of lung bioengineering is probably limited since there are no evidences that MSC can differentiate into different types of lung cells. In fact, it has been suggested that the role of these adult stem cells in lung bioengineering could be to remodel the extracellular matrix of the scaffold for providing a better microenvironment to other infused cells capable of differentiation into lung phenotypes .Given the potential effects induced by physical stimuli on stem cells within the lung, it is expected that optimizing the bioengineering process would require a bioreactor capable of applying ventilation, perfusion and oxygenation conditions facilitating migration, cell-ECM adhesion, proliferation and differentiation of the stem cells seeded into an acellular lung scaffold.ex vivo conditions [Lung bioreactors should include general characteristics of other organ bioreactors , much of them in common with conventional settings to maintain lungs under nditions . Moreovenditions , 85.Lung bioengineering studies performed in the last years have described a variety of methods and protocols for cell seeding and culturing into a lung scaffold, making it difficult to compare the reported results –14. ThesBonvillain et al.  adapted A remarkable study by Ren et al.  focused Improving our understanding of the mechanobiology of stem cells should help in optimizing the ventilation and perfusion settings of the bioreactor for lung bioengineering. Indeed, better understanding the effect of the air-liquid interface on cell homing and differentiation could provide clues on selecting air or liquid ventilation at the different stages of lung biofabrication. Moreover, insight into the role played by compression in different cell types may suggest whether negative pleural pressure ventilation is preferable to positive airway pressure ventilation. Furthermore, better understanding how static and dynamic stretch modulate stem cell fate should be useful for determining the optimal frequencies and amplitudes of ventilation, the most suitable lung inflation volume at end expiration and whether continuous or pulsatile perfusion is more adequate.Given the role that physical stimuli play in stem cell fate, using optimized bioreactors providing stimuli adapted to lung physiology (ventilation and perfusion) is expected to be crucial for the progress of lung bioengineering. Current works in this field of regenerative medicine go beyond research using lung slices and are carried out in whole organs within complex bioreactors, thus providing useful information and anticipating more scientific progress. However, although it has considerably progressed in the last years, the field of lung bioengineering is still in its infancy. Accordingly, the information available is scarce and difficult to interpret, in particular because some findings have been reported by just one or few laboratories and hence ample reproducibility is still lacking. Also, some methodologies should be improved and more widely used, for instance those characterizing the composition and immunogenicity of decellularized lungs and those adequately identifying the phenotype of differentiated cells. More generally, there are still key questions that need to be clarified and much better understood. Specifically, which are the most suitable cell types, media and growth factors (being probably different for the airway and vascular compartments), and how to provide the optimal conditions of ventilation, perfusion and oxygenation to the lung along the process of biofabrication. Advancing into the solution of these open issues requires interdisciplinary research joining stem cell biologists, lung physiologists and experts in biophysics and tissue engineering methodologies.


Barnes et al., in this special issue, tested the hypothesis that more effective photoprotective mechanisms may explain the greater upward migration potential of exotic compared to native species. After surveying the leaf epidermal UV-A-transmittance (inversely related to the accumulation of flavonoids and related UV-absorbing compounds) in 54 species, the authors found that plasticity in this parameter poorly explained the different migration potential of native and exotic species, despite significant intra-specific differences. Rull and Vegas-Vilarrubia also present a remarkable case study of the impact of barriers on plant migration. Authors draw the attention on “The Lost World,” the neotropical Guayana highlands. This pristine habitat, which never suffered from direct human pressure, has been recently exposed to the indirect pressure of human activities, such as global warming and altered rainfall regime. Migration toward higher altitudes is prevented in this habitat because of the flat tops of the highlands. Species with higher phenotypic and physiological plasticity are better suited to survive the challenges associated to climate change. The relative significance of environmental/ and “spatial” (barrier) factors on vegetation dynamics has been explored, at large level of scale, by Chen et al. Authors give compelling evidence that environmental factors are primary drivers of shrub species turnover, whereas barriers mostly hamper the turnover of trees. Wang Z. et al. have also explored traits, such as the germination potential, responsible for effective species dispersal. It is shown that this trait is under tight control of environmental pressures, on short-term scale level, whereas phylogenic constrains become prevalent on long-term basis.Multiple environmental stressors, on both short-term and long-term basis, challenged early land plants since they colonized the land, around 450 million years ago. Emerged lands were far more sensitive to changes in climate than oceans, and plants have displayed during evolution an extraordinary ability to survive  dramatic variations in environmental conditions. This has occurred through acclimation, adaptation, or migration to new environments : de la Riva et al. provided rationale of such biodiversity, and highlighted that different biogeochemical niche partitioning allows species to coexist through divergent leaf nutrient composition and resource uptake.It is not surprising to observe that plants, due to their sessile nature and their slow migration speed, have developed a complex suite of modular and highly integrated mechanisms to match the key constrains encountered within the species distribution range, broadly the acclimation/adaptation syndrome  pest outbreak, and human disturbance, often occur simultaneously and interact with each other. While literature is full of experiments evaluating plant response to single stressors, research investigating the interaction among co-occurring stressors is scarce , which may determine a change of dominant species in plant communities. Ozone is a secondary gaseous pollutant markedly influencing plant communities. Cotrozzi et al. explored the interaction between ozone and drought stresses on the evergreen broad-leafed Quercus ilex. It is shown, that plants challenged by water deficit display decreased capacity to activate phytohormone-related signaling pathways, and hence proper warning signals, when successively exposed to acute O3 stress. Ozone is formed in the troposphere when nitrogen oxide reacts with volatile organic compounds (VOC). A fraction of total VOCs in the atmosphere has biogenic origin  and primarily emitted by plants. After reviewing why biogenic isoprene emission may become relevant in a warmer and drier climate, Fini et al. focused on its metabolic roles, with particular reference to hygrophilic species. Most isoprene emitters are hygrophylic and display a constitutive suite of morpho-anatomical and physiological features unable to efficiently counter the challenges imposed by harsh climates  giving new evidence that may help to better understand how plants will cope with stress factors and novel pressures imposed by the global warming and anthropogenic pressure. However, this topic is wide, which and has been highlighted in other research topics in Frontiers in Plant Science, namely “water stress responses in plants,” “mechanisms of abiotic stress responses and tolerance in plants,” or “photosynthesis within changing climate conditions.” These three are only examples of the 90 research topics published regarding “climate change” or of the 450 concerning “plant responses to stress.” Nonetheless, further experimentation is urgently needed to understand and predict the interactions that underpin the special capacity of plants to fine-tune their physiology and (secondary) metabolism to successfully respond to the severe challenges imposed by a rapidly changing climate, particularly in most fragile ecosystems worldwide. This requires approaches at different levels of scale, using bottom-up as well as top-down approaches, not only to breed “resistant” crops and/or selected varieties, but also to additionally develop strategies for promoting both biodiversity conservation and ecosystem functionality.Understanding plant response to the unprecedented challenges associated to rapid climate change has been the aim of this Research Topic. Such knowledge is urgently needed to develop effective conservation strategies, and to set-up long-term monitoring networks aimed at observing the impact of multiple stress pressures on forest ecosystems. Suitable physiological indicators for this purpose, such as carbon isotope composition and photosystem II photochemistry efficiency, are well described in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Total knee replacement (TKR) has been performed for patients with end-stage knee joint arthritis to relieve pain and gain functions. Most knee replacement patients can gain satisfactory knee functions; however, the range of motion of the implanted knee is variable. There are many designs of TKR implants; it has been suggested by some researchers that customized implants could offer a better option for patients. Currently, the 3-dimensional knee model of a patient can be created from magnetic resonance imaging (MRI) or computed tomography (CT) data using image processing techniques. The knee models can be used for patient-specific implant design, biomechanical analysis, and creating bone cutting guide blocks. Researchers have developed patient-specific musculoskeletal lower limb model with total knee replacement, and the models can be used to predict muscle forces, joint forces on knee condyles, and wear of tibial polyethylene insert. These available techniques make it feasible to create customized implants for individual patients. Methods and a workflow of creating a customized total knee replacement implant for improving TKR kinematics and functions are discussed and presented in this paper. Total knee replacement (TKR) has been widely used to relieve osteoarthritis pain, and it has been established as a successful treatment for advanced degenerative joint disease. TKR is expected to rise due to the aging population, obesity, and public expectations. A typical TKR implant has a metal femoral component, a metal tibial tray, a polyethylene insert, and a polyethylene button. One of the main aims of TKR is for a patient to walk postoperatively; however, Milner  showed tIn the past decades, there have been attempts to create a more natural feeling and anatomical TKR. The objective of this paper was to review the latest development on TKR, then propose an approach to making customized total knee replacement implants which can function as close as possible to the normal knee of the patient.Literature review was conducted related to TKR using PUBMED database . There are 336 papers available through a PUBMED search  using the query “Total knee replacement/arthroplasty” and “patient specific instrumentation.” However, there are 68 papers available through a PUBMED search  using the query “Total knee replacement/arthroplasty” and “customized.” Among these 68 papers, 28 papers are relevant to the knee replacement and patient-specific instrumentation (PSI) or implant designs. There were recent review papers on PSI by Rodrigues and Gutierres [There are many TKR implants available in the market; each of them has its design rationale. Different designs of implant aim to enhance the satisfaction of patients by providing close to normal kinematics. The femoral condyle in sagittal plane may be circular shaped as shown in The motion of the medial compartment in TKR is normally simplified into a ball-and-socket; however, the lateral converged femoral condyle in a surface-guided knee implant has been designed to control the motion of the joint. The lateral condyle may be designed to produce a constant or variable bearing distance between the medial condyle and lateral condyle. To achieve close to normal kinematics in TKR, Walker  showed tKim et al.  comparedWith regard to implant wear, Abdelgaied et al.  investigCuller et al.  comparedMany researches showed that women had significantly narrower distal femoral condyle width than men. Wise et al.  showed tL1 in the sagittal plane; this curve is a condyle profile that matches a patient's knee shape. The circular curve and the other curve L2 are created based on a patient knee model. The curve L2 sweeps along the curve L1 from point A to point B and creates a condylar articular surface. BS is the distance between two condyle profiles, and it is determined from a patient knee model.Customized TKR implant has been developed based on the patient's MRI/CT data. Carr and Goswami  reviewedLower limb mechanical axis restoration is very important for long-term survivorship of TKR. Recently, patient-specific instrumentation or patient-specific cutting block/guide has been developed to help improve mechanical axis alignment. Medical images can be processed to create 3D models, along with the development of 3D printing technologies. There has been an increased use of 3D printing techniques in patient-specific treatments. 3D printing can be used mostly to create patient-specific anatomical models, customized moulds, surgical guides, and implants. It has been reported that patient-specific guide or cutting block can provide guidance to surgeons during surgery, and this can minimize tissue loss and optimize the positioning of implants. A distal femoral cutting guide is shown in Both MRI and CT imaging have been used for creation of patient-specific guides. MRI is able to account for residual articular cartilage; therefore, the cutting guide can cover a broad contact area and can be directly placed on bone and residual cartilage of knee joint. CT is unable to account for residual cartilage; the corresponding cutting guide has to rely on multiple bony sites. Frye et al.  concludePatient knee shapes are well known to be different; the surface geometry of TKR implant affects joint congruence and contact mechanics. It has been suggested by Pati et al.  that cusGoyal and Tripathy  have surHowever, Rodrigues and Gutierres  reviewedPark et al.  investigMusculoskeletal models can be created using software such as AnyBody and Opensim. A lower limb musculoskeletal model with a TKR implant is shown in Knowledge of muscle and joint loading is important for evaluating the performance of TKR implant. If the knee is assumed as a pin joint in MSK models, it will produce erroneous results in knee muscle forces and moments acting in the frontal and transverse planes. Walter and Pandy  simulateLower limb musculoskeletal model has been used in the analysis of joint mechanics and kinematics. Many researchers have tried different methods to create musculoskeletal models. Knarr and Higginson  proposedMusculoskeletal models can be used for implant wear analysis. TKR implant has traditionally been tested in knee wear simulator to determine its ability to resist wear. The computational models can be used to predict wear of implant as did by Zhang et al. , and thePatient's gait dataset was used in the modelling, and this may raise the question of obtaining the gait data. The evolution of the knee gait kinematics in patients with knee osteoarthritis before and several months after a total knee replacement has been researched and presented by Bonnefoy-Mazure et al.  and RahmBased on the progress described in the previous sections, a new approach which is different from the current customized knee implant design is proposed in this paper. The focus of this approach is using patient-specific loading and gait dataset for knee implant designs in addition to the knee joint anatomical features. The procedure of creating a customized TKR implant is shown in To design a customized TKR implant, 3D computer models from CT scans or MRI should be created firstly. The gait and foot reaction forces of the patient will be measured preoperatively and used for the prediction of load and kinematics of knee joint. Postoperative gait characteristics of patient can be predicted using the derived relationship between measured preoperative and postoperative gait datasets; then, the predicted patient gait is used in the MSK modelling. The interaction between TKR implant and knee joint dynamics will be evaluated using MSK models. The optimal TKR implant should reproduce knee function, maintain bone-implant interface integrity, and resist wear. The kinematics and loads on the TKR implant are very important to the success of TKR. To create a customized TKR implant, an iteration procedure is required to optimise stress, material wear, and knee kinematics.Customized TKR implant has the potential to greatly improve knee kinematics and patient knee functions compared to off-the-shelf TKR implant. However, further studies need to be carried out to make the customized TKR implant available for patients.Customized total knee replacement implant has been previously designed considering knee anatomical shape; however, with the latest development on lower limb musculoskeletal models, force dependent kinematics, and wear simulations, a customized total knee replacement implant could be developed to enhance patient satisfaction. The workflow of the approach to making customized TKR implant is presented in this paper. The customized total knee replacement implant will not only replicate the shape of the knee joint but also to restore normal gait of the patient postoperatively.


We identified a ΔpyrE mutant as under selection in screening of a Salmonella mutant library in 4-day old chicks. Here, we confirm that a ΔpyrE mutant colonizes 4-day old chicks poorly in competitive infection with isogenic wild type, and that the ability of this mutant to colonize chicks could be restored by providing a copy of pyrE in trans. We further show that our ΔpyrE mutant grows poorly in nutrient poor conditions in vitro, and that the ability of this mutant to grow is restored, both in vitro and in chicks, when precursors to the pyrimidine salvage pathway were provided. This finding suggests that the environment in the chick intestine during our infections lacks sufficient precursors of the pyrimidine salvage pathway to support Salmonella growth. Finally, we show that the colonization defect of a ΔpyrE mutant during infection occurs in to chicks, but not in CBA/J mice or ligated ileal loops in calves. Our data suggest that de novo pyrimidine synthesis is necessary for colonization of Salmonella Typhimurium in the chick, and that the salvage pathway is not used in this niche. De novo pyrimidine synthesis occurs in all organisms . Li. LiSalmopyrE gene as a candidate needed for colonization and persistence of Salmonella Typhimurium after oral infection of 4-day old chicks. pyrE encodes orotate phosphoribosyltransferase, the fifth enzyme involved in pyrimidine biosynthesis. PyrE catalyzes the formation of orotidine 5’-monophosphate (OMP) from orotate and phosphoribosyl pyrophosphate (pyrE (STM3733) is shown in We identified the hosphate  14]. Th. ThpyrE pyrE during colonization and persistence in this animal model, we retested the ΔpyrE mutant in chicks in competitive infection with isogenic wild type, HA431 (ATCC14028 ΔphoN::KanR). A mutant in pyrE colonized all organs tested poorly from the earliest points in infection. The pyrE null mutant had a significant reduction in colonization of all organs that was most severe in the gastrointestinal tract with greater than a 1000-fold reduction relative to the wild type at day 3 post infection , and monitored shedding of these strains in feces to 41 days post-infection  or regular (uracil -) diets, respectively. At day 4 post-hatch, both groups of chicks were infected with equal mixtures of ΔpyrE mutant and isogenic wild type, HA431.A previous study has reported that the enzyme activity of carbamyl phosphate synthetase  and aspartate transcarbamylase , the first 2 enzymes in  pigeons . We hypoSalmonella colonization. At this point, remaining chicks from the uracil-supplemented group were subdivided into 2 groups. The first group was maintained on uracil-supplemented food (uracil +/+) while the second group had uracil withdrawn from their diets (uracil +/-). At day 9 post-infection, these remaining chicks were euthanized and organs were collected and processed to compare Salmonella colonization between groups . As expected, the nfection . Moreovede novo synthesis of pyrimidines from small molecules, or by a salvage pathway that utilizes preformed pyrimidine bases or nucleosides. The de novo pyrimidine biosynthetic pathway is highly conserved between animals and microorganisms [Salmonella Typhimurium [de novo pyrimidine synthesis in Salmonella is critical for this organism to colonize the intestine in chicks.The synthesis of pyrimidine nucleotides occurs through two distinct pathways: via pathway for rganisms  and it ihimurium . Both a himurium . We now pyrE, which behaves as a pyrimidine auxotroph in vitro, has a severe colonization defect in this chicks. The growth defect of our ΔpyrE mutant in minimal media is reversed when uracil is provided and the salvage pathway can be used instead , the fourth enzyme in the pyrimidine biosynthetic pathway encoding dihydroorotate dehydrogenase , also grow poorly in pyrimidine limited conditions [pyrC, the third enzyme in the pyrimidine biosynthetic pathway   , is up-rhimurium . Our finpyrE deletion mutant colonized chicks very poorly, it appears to colonize ligated ileal loops in calves and orally infected CBA/J mice very similar to the wild type organism. The activities of pyrA and pyrB, encoding carbamyl phosphate synthetase  and aspartate transcarbamylase , respectively, differ in various animals including rat, mice, pigeon and chick but are especially low in 3-week old chicks compared to other animal models, supporting our findings [pyrE mutants in Listeria monocytogenes also indicated that pyrE is not involved in virulence and colonization of Listeria in C3H and BALB/c mice [Although our findings . AdditioB/c mice . These fΔpyrE mutant was rescued by the addition of uracil to the chick diets over the 9-day infections we performed. While the ΔpyrE mutant colonized similar to the wild type when the diet was supplemented with uracil, uracil deprivation after the establishment of infection resulted in the immediate loss of the ability of the ΔpyrE mutant to colonize the intestinal tract. However, withdrawal of uracil supplementation after the establishment of infection in the presence of uracil did not affect the ability of the pyrE mutant to colonize and persist in the cecal tonsil and bursa. These results are in agreement with previous findings that a ΔpyrE mutant in Listeria monocytogenes is pyrimidine auxotroph, and uracil supplementation restores the growth of this mutant to wild type levels [Finally, the colonization defect of our e levels .ΔpyrE mutant in Salmonella Typhimurium does not grow in pyrimidine-limited media, and poorly colonized in 4-day old chicks. Reduced growth of the ΔpyrE mutant in pyrimidine-limited conditions could be restored through addition of uracil, which can be utilized in a salvage pathway to generate pyrimidines. Moreover, it appears that pyrimidine biosynthesis is not limiting to Salmonella growth during infection of every animal model, suggesting that one could use auxotrophies to probe the nutritional environment of different niches during infection.To summarize, we show that a S1 File(XLSX)Click here for additional data file.