Inline dynamometry provides reliable measurements of quadriceps strength in healthy and ACL-reconstructed individuals and is a valid substitute for isometric electromechanical dynamometry following ACL reconstruction.

BACKGROUND: Quadriceps strength testing is recommended to guide rehabilitation and mitigate the risk of second injury following anterior cruciate ligament (ACL) reconstruction. Hand-held dynamometry is a practical alternative to electromechanical dynamometry but demonstrates insufficient reliability and criterion validity in healthy and ACL-reconstructed participants respectively. The purpose of this study is to investigate the reliability and concurrent validity of inline dynamometry for measuring quadriceps strength. The hypotheses are that intra-class correlation coefficient (ICC) values will be >0.90 for reliability and concurrent validity. METHODS: This was a cross sectional study using a within-participant, repeated measures design. Isometric quadriceps testing was performed at 60° knee flexion in 50 healthy and 52 ACL-reconstructed participants. Interrater reliability, intrarater reliability, and concurrent validity of inline dynamometry was investigated through calculation of ICCs, Bland-Altman analysis, linear regression, standard error of measurement (SEM) and minimal detectable change (MDC). RESULTS: The lower bounds of the 95% confidence intervals were >0.90 for all reliability and validity ICCs in healthy and ACL-reconstructed participants, except for intrarater reliability in healthy participants using absolute scores (ICC = 0.936 [95% CI 0.890-0.963]). In ACL-reconstructed participants, Bland-Altman bias was 0.01 Nm/kg for absolute and average scores, limits of agreement were -11.74% to 12.59% for absolute scores, the SEM was 0.13Nm/kg (95% CI 0.10-0.17) and the MDC was 0.36Nm/kg (95% CI 0.28 - 0.47). CONCLUSION: Inline dynamometry is a reliable and economical alternative to electromechanical dynamometry for the assessment of quadriceps strength following ACL-reconstruction. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT05109871).

Mesenchymal Stem Cells in the Pathogenesis and Therapy of Autoimmune and Autoinflammatory Diseases.

Mesenchymal stem cells (MSCs) modulate immune responses and maintain self-tolerance. Their trophic activities and regenerative properties make them potential immunosuppressants for treating autoimmune and autoinflammatory diseases. MSCs are drawn to sites of injury and inflammation where they can both reduce inflammation and contribute to tissue regeneration. An increased understanding of the role of MSCs in the development and progression of autoimmune disorders has revealed that MSCs are passive targets in the inflammatory process, becoming impaired by it and exhibiting loss of immunomodulatory activity. MSCs have been considered as potential novel cell therapies for severe autoimmune and autoinflammatory diseases, which at present have only disease modifying rather than curative treatment options. MSCs are emerging as potential therapies for severe autoimmune and autoinflammatory diseases. Clinical application of MSCs in rare cases of severe disease in which other existing treatment modalities have failed, have demonstrated potential use in treating multiple diseases, including rheumatoid arthritis, systemic lupus erythematosus, myocardial infarction, liver cirrhosis, spinal cord injury, multiple sclerosis, and COVID-19 pneumonia. This review explores the biological mechanisms behind the role of MSCs in autoimmune and autoinflammatory diseases. It also covers their immunomodulatory capabilities, potential therapeutic applications, and the challenges and risks associated with MSC therapy.

Cardiac Mesenchymal Stem Cell-like Cells Derived from a Young Patient with Bicuspid Aortic Valve Disease Have a Prematurely Aged Phenotype.

There is significant interest in the role of stem cells in cardiac regeneration, and yet little is known about how cardiac disease progression affects native cardiac stem cells in the human heart. In this brief report, cardiac mesenchymal stem cell-like cells (CMSCLC) from the right atria of a 21-year-old female patient with a bicuspid aortic valve and aortic stenosis (referred to as biscuspid aortic valve disease BAVD-CMSCLC), were compared with those of a 78-year-old female patient undergoing coronary artery bypass surgery (referred to as coronary artery disease CAD-CMSCLC). Cells were analyzed for expression of MSC markers, ability to form CFU-Fs, metabolic activity, cell cycle kinetics, expression of NANOG and p16, and telomere length. The cardiac-derived cells expressed MSC markers and were able to form CFU-Fs, with higher rate of formation in CAD-CMSCLCs. BAVD-CMSCLCs did not display normal MSC morphology, had a much lower cell doubling rate, and were less metabolically active than CAD-CMSCLCs. Cell cycle analysis revealed a population of BAVD-CMSCLC in G2/M phase, whereas the bulk of CAD-CMSCLC were in the G0/G1 phase. BAVD-CMSCLC had lower expression of NANOG and shorter telomere lengths, but higher expression of p16 compared with the CAD-CMSCLC. In conclusion, BAVD-CMSCLC have a prematurely aged phenotype compared with CAD-CMSCLC, despite originating from a younger patient.

1H NMR Metabolite Monitoring during the Differentiation of Human Induced Pluripotent Stem Cells Provides New Insights into the Molecular Events That Regulate Embryonic Chondrogenesis.

The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used 1H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that 1H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.

The diagnostic accuracy of clinical tests for anterior cruciate ligament tears are comparable but the Lachman test has been previously overestimated: a systematic review and meta-analysis.

PURPOSE: The diagnostic accuracy of clinical tests for anterior cruciate ligament injury has been reported in previous systematic reviews. Numerous studies in these reviews include subjects with additional knee ligament injury, which could affect the sensitivity of the tests. Meta-analyses have also been performed using methods that do not account for the non-independence of sensitivity and specificity, potentially overestimating diagnostic accuracy. The aim of this study was to report the diagnostic accuracy of clinical tests for anterior cruciate ligament tears (partial and complete) without concomitant knee ligament injury. METHODS: A systematic review with meta-analysis was performed according to the PRISMA guidelines. Meta-analyses included studies reporting the specificity and/or sensitivity of tests with or without concomitant meniscal injury. Where possible, pooled diagnostic estimates were calculated with bivariate random-effects modelling to determine the most accurate effect sizes. Diagnostic accuracy values are presented for the anterior drawer, Lachman, Lever sign and pivot shift tests overall and in acute or post-acute presentations. RESULTS: Pooled estimates using a bivariate model for overall sensitivity and specificity respectively were as follows: anterior drawer test 83% [95% CI, 77-88] and 85% [95% CI, 64-95]; Lachman test 81% [95% CI, 73-87] and 85% [95% CI, 73-92]; pivot shift test 55% [95% CI, 47-62] and 94% [95% CI, 88-97]; Lever sign test 83% [95% CI, 68-92] and 91% [95% CI, 83-95]. For specific presentations, the sensitivity and specificity of the Lachman test, respectively, were: complete tears 68% [95% CI, 54-79] and 79% [95% CI, 51-93]; post-acute injuries 70% [95% CI, 57-80] and 77% [95% CI, 53-91]. CONCLUSIONS: The pivot shift and Lever sign were the best tests overall for ruling in or ruling out an anterior cruciate ligament tear, respectively. The diagnostic accuracy of the Lachman test, particularly in post-acute presentations and for complete tears, is lower than previously reported. Further research is required to establish more accurate estimates for the Lachman test in acute presentations and partial ligament tears using bivariate analysis. LEVEL OF EVIDENCE: III.

Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control?

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM's structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

Juvenile idiopathic arthritis: from aetiopathogenesis to therapeutic approaches.

Juvenile idiopathic arthritis (JIA) is the most common paediatric rheumatological disorder and is classified by subtype according to International League of Associations for Rheumatology criteria. Depending on the number of joints affected, presence of extra-articular manifestations, systemic symptoms, serology and genetic factors, JIA is divided into oligoarticular, polyarticular, systemic, psoriatic, enthesitis-related and undifferentiated arthritis. This review provides an overview of advances in understanding of JIA pathogenesis focusing on aetiology, histopathology, immunological changes associated with disease activity, and best treatment options. Greater understanding of JIA as a collective of complex inflammatory diseases is discussed within the context of therapeutic interventions, including traditional non-biologic and up-to-date biologic disease-modifying anti-rheumatic drugs. Whilst the advent of advanced therapeutics has improved clinical outcomes, a considerable number of patients remain unresponsive to treatment, emphasising the need for further understanding of disease progression and remission to support stratification of patients to treatment pathways.

Investigating the biological response of human mesenchymal stem cells to titanium surfaces.

BACKGROUND: We have investigated the behaviour of a newly characterised population of haemarthrosis fluid-derived human mesenchymal stem cells (HF-hMSCs) with titanium (Ti) surfaces. METHODS: HF-hMSCs were seeded onto round cannulated interference (RCI; Smith and Nephew) screws or control Ti discs and cultured under pro-osteogenic conditions. RESULTS: Electron microscopy showed the attachment and spreading of HF-hMSCs across both Ti surfaces during the early stages of osteogenic culture; however, cells were exclusively localised to the basal regions within the vertex of the Ti screws. In the later stages of culture, an osteoid matrix was deposited on the Ti surfaces with progressive culture expansion and matrix deposition up the sides and the top of the Ti Screws. Quantification of cellular content revealed a significantly higher number of cells within the Ti screw cultures; however, there was no difference in the cellular health. Conversely, alizarin red staining used as both a qualitative and quantitative measure of matrix calcification was significantly increased in Ti disc cultures compared to those of Ti screws. CONCLUSIONS: Our results suggest that the gross topography of the metal implant is able to create microenvironment niches that have an influence on cellular behaviour. These results have implications for the design of advanced tissue engineering strategies that seek to use cellular material to enhance biological remodelling and healing following tissue reconstruction.

Biocompatibility and enhanced osteogenic differentiation of human mesenchymal stem cells in response to surface engineered poly(D,L-lactic-co-glycolic acid) microparticles.

Tissue engineering strategies can be applied to enhancing osseous integration of soft tissue grafts during ligament reconstruction. Ligament rupture results in a hemarthrosis, an acute intra-articular bleed rich in osteogenic human mesenchymal stem cells (hMSCs). With the aim of identifying an appropriate biomaterial with which to combine hemarthrosis fluid-derived hMSCs (HF-hMSCs) for therapeutic application, this work has investigated the biocompatibility of microparticles manufactured from two forms of poly(D,L-lactic-co-glycolic acid) (PLGA), one synthesized with equal monomeric ratios of lactic acid to glycolic acid (PLGA 50:50) and the other with a higher proportion of lactic acid (PLGA 85:15) which confers a longer biodegradation time. The surfaces of both types of microparticles were functionalized by plasma polymerization with allylamine to increase hydrophilicity and promote cell attachment. HF-hMSCs attached to and spread along the surface of both forms of PLGA microparticle. The osteogenic response of HF-hMSCs was enhanced when cultured with PLGA compared with control cultures differentiated on tissue culture plastic and this was independent of the type of polymer used. We have demonstrated that surface engineered PLGA microparticles are an appropriate biomaterial for combining with HF-hMSCs and the selection of PLGA is relevant only when considering the biodegradation time for each biomedical application.

Low oxygen tension is critical for the culture of human mesenchymal stem cells with strong osteogenic potential from haemarthrosis fluid.

Satisfactory osseous tissue integration of the soft tissue graft with bone is the mainstay of healing following surgical reconstruction of the anterior cruciate ligament (ACL). However, tissue remodelling is slow and significantly impacts on quality of life by delaying return to work and sport and accelerating the onset of degenerative diseases such as osteoarthritis. Delivery of multipotent human mesenchymal stem cells (hMSCs) at surgery could enhance osseous tissue integration. We aim to use hMSCs derived from haemarthrosis fluid (HF) (the intra-articular bleed accrued post-trauma) which is aspirated and discarded as clinical waste. With the aim of improving our bioprocessing methodologies for clinical translation we have investigated the effect of low oxygen tension on the derivation and osteogenic potential of this novel HF-hMSC population. Mononuclear cells were isolated from HF aspirated samples and divided for derivation and culture under normal or low oxygen tension. HF-hMSCs were derived from 100 % of cultures under low oxygen tension compared to 71 % for normal oxygen tension; this was coupled with increased CFU-Fs. We investigated the osteogenic potential and cellular health of HF-hMSC populations following ex vivo expansion. HF-hMSC populations showed enhanced matrix mineralisation and cellular health when differentiated under low oxygen tension. This positive effect of low oxygen on osteogenesis and cellular health was reduced with prolonged culture. These data demonstrate that derivation and culture of HF-hMSC populations under low oxygen tension will enable the translation of a cellular therapy for the treatment of broad patient numbers with optimal osteogenic potency and cellular vitality.

Cell sources for the regeneration of articular cartilage: the past, the horizon and the future.

Avascular, aneural articular cartilage has a low capacity for self-repair and as a consequence is highly susceptible to degradative diseases such as osteoarthritis. Thus the development of cell-based therapies that repair focal defects in otherwise healthy articular cartilage is an important research target, aiming both to delay the onset of degradative diseases and to decrease the need for joint replacement surgery. This review will discuss the cell sources which are currently being investigated for the generation of chondrogenic cells. Autologous chondrocyte implantation using chondrocytes expanded ex vivo was the first chondrogenic cellular therapy to be used clinically. However, limitations in expansion potential have led to the investigation of adult mesenchymal stem cells as an alternative cell source and these therapies are beginning to enter clinical trials. The chondrogenic potential of human embryonic stem cells will also be discussed as a developmentally relevant cell source, which has the potential to generate chondrocytes with phenotype closer to that of articular cartilage. The clinical application of these chondrogenic cells is much further away as protocols and tissue engineering strategies require additional optimization. The efficacy of these cell types in the regeneration of articular cartilage tissue that is capable of withstanding biomechanical loading will be evaluated according to the developing regulatory framework to determine the most appropriate cellular therapy for adoption across an expanding patient population.

Directed differentiation of human embryonic stem cells toward chondrocytes.

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

Derivation of Man-1 and Man-2 research grade human embryonic stem cell lines.

We report here the derivation of two new human embryonic stem cell lines, Man-1 and Man-2, and their full characterization as novel pluripotent stem cell lines. Man-1 was derived from an embryo surplus to requirement from routine IVF, while Man-2 was obtained from an oocyte classified as failed to fertilise and subsequently chemically activated. We report the characterisation of pluripotency and the differentiation potential of these lines. Work is in progress to establish novel methods of stem cell derivation and culture, which will avoid the use of xenobiotics and be relevant to clinical production of human embryonic stem cell lines. Both newly derived human embryonic stem cell lines will be available for the research community from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).

Notch signaling during chondrogenesis of human bone marrow stem cells.

Notch signaling is an important mechanism involved in early development which helps to determine the differentiation and fate of cells destined to form different tissues in the body. Its role in the differentiation of adult stem cells, such as those found in bone marrow is much less clear. As there is great interest in the potential of human bone marrow stem cells (hMSC) as a source of cells for the repair of articular cartilage and other tissues, it is important to understand if Notch signaling promotes or suppresses differentiation. Using primary human bone marrow stem cells (hMSC) in 3D cell aggregate culture a new study has investigated the expression of the canonical Notch pathway genes during chondrogenesis and showed that the Notch ligand, Jagged1 (JAG1) sharply increased in expression peaking early in differentiation. A Notch target gene, HEY1, was also expressed with a temporal profile, which closely followed the expression of JAG1 and this preceded the rise in type II collagen expression that characterized chondrogenesis. The JAG1 mediated Notch signaling was shown with a Notch inhibitor (DAPT) to be necessary for chondrogenesis, as inhibition days 0-14, or just days 0-5, blocked chondrogenesis, whereas Notch inhibition days 5-14 did not. In further experiments Notch signaling was shown to be critical for full chondrogenesis, as adenoviral hJAG1 transduction of hMSCs, which caused continuous expression of JAG1 and sustained Notch signaling, completely blocked chondrogenesis. In these cultures there was inhibited production of extracellular matrix and failure to differentiate was interpreted as the retention of the hMSC in a pre-chondrogenic state. The results in this study thus showed that JAG1 mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but must be switched off for chondrogenesis to proceed and it will be important to establish if this is a mechanism common to all chondrocyte differentiation.

Notch signaling through Jagged-1 is necessary to initiate chondrogenesis in human bone marrow stromal cells but must be switched off to complete chondrogenesis.

We investigated Notch signaling during chondrogenesis in human bone marrow stromal cells (hMSC) in three-dimensional cell aggregate culture. Expression analysis of Notch pathway genes in 14-day chondrogenic cultures showed that the Notch ligand Jagged-1 (Jag-1) sharply increased in expression, peaking at day 2, and then declined. A Notch target gene, HEY-1, was also expressed, with a temporal profile that closely followed the expression of Jag-1, and this preceded the rise in type II collagen expression that characterized chondrogenesis. We demonstrated that the shut-down in Notch signaling was critical for full chondrogenesis, as adenoviral human Jag-1 transduction of hMSC, which caused continuous elevated expression of Jag-1 and sustained Notch signaling over 14 days, completely blocked chondrogenesis. In these cultures, there was inhibited production of extracellular matrix, and the gene expression of aggrecan and type II collagen were strongly suppressed; this may reflect the retention of a prechondrogenic state. The JAG-1-mediated Notch signaling was also shown to be necessary for chondrogenesis, as N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) added to cultures on days 0-14 or just days 0-5 inhibited chondrogenesis, but DAPT added from day 5 did not. The results thus showed that Jag-1-mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but it must be switched off for chondrogenesis to proceed.

Cartilage, SOX9 and Notch signals in chondrogenesis.

Cartilage repair is an ongoing medical challenge. Tissue engineered solutions to this problem rely on the availability of appropriately differentiated cells in sufficient numbers. This review discusses the potential of primary human articular chondrocytes and mesenchymal stem cells to fulfil this role. Chondrocytes have been transduced with a retrovirus containing the transcription factor SOX9, which permits a greatly improved response of the cells to three-dimensional culture systems, growth factor stimulation and hypoxic culture conditions. Human mesenchymal stem cells have been differentiated into chondrocytes using well-established methods, and the Notch signalling pathway has been studied in detail to establish its role during this process. Both approaches offer insights into these in vitro systems that are invaluable to understanding and designing future cartilage regeneration strategies.