In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair.

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development.

Precision health aims to develop personalised and proactive strategies for predicting, preventing, and treating complex diseases such as osteoarthritis (OA). Due to OA heterogeneity, which makes developing effective treatments challenging, identifying patients at risk for accelerated disease progression is essential for efficient clinical trial design and new treatment target discovery and development.

To create a reliable and interpretable precision health tool that predicts rapid knee OA progression over a 2-year period from baseline patient characteristics using an advanced automated machine learning (autoML) framework, “Autoprognosis 2.0”.

All available 2-year follow-up periods of 600 patients from the FNIH OA Biomarker Consortium were analysed using “Autoprognosis 2.0” in two separate approaches, with distinct definitions of clinical outcomes: multi-class predictions (categorising disease progression into pain and/or radiographic progression) and binary predictions. Models were developed using a training set of 1352 instances and all available variables (including clinical, X-ray, MRI, and biochemical features), and validated through both stratified 10-fold cross-validation and hold-out validation on a testing set of 339 instances. Model performance was assessed using multiple evaluation metrics. Interpretability analyses were carried out to identify important predictors of progression.

Our final models yielded higher accuracy scores for multi-class predictions (AUC-ROC: 0.858, 95% CI: 0.856-0.860) compared to binary predictions (AUC-ROC: 0.717, 95% CI: 0.712-0.722). Important predictors of rapid disease progression included WOMAC scores and MRI features. Additionally, accurate ML models were developed for predicting OA progression in a subgroup of patients aged 65 or younger.

This study presents a reliable and interpretable precision health tool for predicting rapid knee OA progression. Our models provide accurate predictions and, importantly, allow specific predictors of rapid disease progression to be identified. Furthermore, the transparency and explainability of our methods may facilitate their acceptance by clinicians and patients, enabling effective translation to clinical practice.

Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood.

The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair.

Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee.

The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process.

Tissue repair is believed to rely on tissue-resident progenitor cell populations proliferating, migrating, and undergoing differentiation at the site of injury. During these processes, the crosstalk between mesenchymal stromal/stem cells (MSCs) and macrophages has been shown to play a pivotal role. However, the influence of extracellular matrix (ECM) remodelling in this crosstalk, remains elusive.

Human MSCs cultured on tissue culture plastic (TCP) and encased within fibrin in vitro were treated with/without TNFα and IFNγ. Human monocytes were cocultured with untreated/pretreated MSCs on TCP or within fibrin. After seven days, the conditioned media (CM) were collected. Human chondrocytes were exposed to CM in a migration assay. The impact of TGFβ was assessed by adding an inhibitor (TGFβRi). Cell activity was assessed using RT-qPCR and XL-protein-profiler-array.

Previously, we demonstrated that culturing human MSCs within 3D-environments significantly enhances their immunoregulatory activity in response to pro-inflammatory stimuli. In this study, monocytes were co-cultured with MSCs within fibrin, acquiring a distinct M2-like repair macrophage phenotype in contrast to TCP co-cultures. MSC/macrophage CM characterization using a protein array demonstrated differences in release of several factors, including chemokines, growth factors and ECM components. Chondrocyte migration was significantly reduced in CM from untreated MSC/monocytes co-cultures in fibrin compared to CM of untreated MSCs/monocytes on TCP. This impact on migration was not seen with chondrocytes cultured in CM of monocytes co-cultured with pretreated MSCs in fibrin. The CM of monocytes co-cultured with pretreated MSCs in fibrin up-regulates COL2A1 and SOX9 compared to TCP. Chondrogenesis and migration were TGFβ dependent.

MSC/macrophage crosstalk and responsiveness to cytokines are influenced by the ECM environment, which subsequently impacts tissue-resident cell migration and chondrogenesis. The direct effects of ECM on MSC/macrophage secretory phenotype is complemented by the dynamic ECM binding and release of growth factors such as TGFβ.

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Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties.

We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Clear differences between the younger and older patients were indicated.

Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes.

Osteochondral injuries are a recognised factor in the development of osteoarthritis (OA). Mesenchymal stromal cells (MSCs) represent a promising biological therapeutic option as an OA-modifying treatment, and they also secrete factors that may have an anti-catabolic effect and/or encourage endogenous repair. We aim to study the effects of (i) intra-articular injection of human bone-marrow-derived MSCs and (ii) their secretome on recovery in a murine knee osteochondral injury model.

The MSC secretome was generated by stimulating human bone-marrow-derived MSCs with tumour necrosis factor alpha (TNFα). Mice (n=48) were injected with i) MSC secretome, ii) MSCs or iii) cell culture medium (control). Pain was assessed by activity monitoring, and cartilage repair, subchondral bone volume and synovial inflammation were evaluated using histology and microCT.

Both MSC- and MSC-secretome-injected mice showed significant pain reduction at day 7 when compared to control mice, but only the MSC-injected mice maintained a significant improvement over the controls at day 28. Cartilage repair was significantly improved in MSC-injected mice. No significant effects were observed with regards to synovial inflammation or subchondral bone volume.

The MSC secretome demonstrates regenerative effects but this does not appear to be as sustained as a MSC cell therapy. Further studies are required to investigate if this can be overcome using different dosing regiments for injection of the MSC secretome. As we further understand the regenerative properties of the MSC secretome, we may be able to enhance the clinical translatability of these therapies. Direct intra-articular injection of MSCs for the treatment of OA also appears promising as a potential future strategy for OA management.

Acknowledgements: MS is supported by a grant from the Wellcome Trust (PhD Programme for Clinicians)

Abstract

Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the infrapatellar fat pad using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes.

Abstract

Focal articular cartilage defects do not heal and, left untreated, progress to more widespread degenerative changes. A promising new approach for the repair of articular cartilage defects is the application of cell-based regenerative therapies using mesenchymal stromal cells (MSCs). MSCs are however present in a number of tissues and studies suggest that they vary in their proliferation, cell surface characterisation and differentiation. As the phenotypic properties of MSCs vary depending on tissue source, a systematic comparison of the transcriptomic signature would allow a better understanding of these differences between tissues, and allow the identification of markers specific to a MSC source that is best suited for clinical application. Tissue was used from patients undergoing total knee replacement surgery for osteoarthritis following ethical approval and informed consent. MSCs were isolated from bone, cartilage, synovium and infrapatellar fat pad. MSC number and expansion were quantified. Following expansion in culture, MSCs were characterised using flow cytometry with several cell surface markers; the cells from all sources were positive for CD44, CD90 and CD105. Their differentiation potential was assessed through tri-lineage differentiation assays. In addition, bulk mRNA-sequencing was used to determine the transcriptomic signatures. Differentially expressed (DE) genes were predicted. An enrichment analysis focused on the DE genes, against GO and pathway databases (KEGG and Reactome) was performed; protein-protein interaction networks were also inferred (Metascape, Reactome, Cytoscape). Optimal sourcing of MSCs will amplify their cartilage regeneration potential. This is imperative for assessing future therapeutic transplantation to maximise the chance of successful cartilage repair. A better understanding of differences in MSCs from various sources has implications beyond cartilage repair.

Osteoarthritis (OA) affects millions of people and is the fastest growing cause of disability worldwide. In order to address this burden, early intervention strategies have been proposed. Therapies that utilise bone marrow stromal cells (BM-MSCs) to induce cartilage repair, either as a cell therapy or by endogenous release by drilling or microfracture, have proved promising. However, limitations include fibrotic features of the regenerated cartilage that may affect mechanical properties and therefore the longevity of such a repair. In order to improve this regenerative technique, further research is required to understand the key players in the repair mechanism. An interaction, which may be important, is that between BM-MSCs and the resident chondrocytes. The aim of this study is to understand the interplay between BM-MSC and resident chondrocytesisolated from different zonal locations within the human knee.

We compared chondrocytes from three different cartilage areas: chondrocytes from 1) the superficial zone (SZ) and 2) the middle-deep (MDZ) zone of non-weight bearing femoral condyles, and from 3) the osteoarthritic zone (OAZ) of patients undergoing knee replacement. First, we evaluated the influence of different chondrocytes on BM-MSCs monolayer in a transwell co-culture, assessing transcript levels of early chondrogenic markers including Sox9 and Col1. Secondly, in a 3D co-culture system, we evaluated how cartilage chips from the three different zones affect the chondrogenic differentiation of BM-MSC pellets. Results indicated that cells from the SZ induce chondrogenic differentiation of BM-MSCs when co-cultured. In contrast, MDZ and OAZ have a negative effect, compared to control conditions. Our findings suggest that chondrocytes from the SZ, a zone which has been reported to reduce with age and may be lost in advanced OA, is important to direct BM-MSCs differentiation towards the chondrogenic fate. This may be relevant to cartilage repair strategies.

Osteoarthritis is characterised by the loss and damage of cartilage in synovial joints. Whilst joint replacement is the gold standard for end stage disease, repair or regenerative strategies aim to slow disease progression, maintain joint function and defer the need for joint replacement. One approach seeks to target endogenous repair after drilling or microfracture (a type of trauma induced repair) in the area of cartilage loss – connecting the defect to the underlying bone marrow niche. The rationale of this approach is that cells delivered to the defect site, from the bone marrow, will bring about cartilage repair.

Bone marrow contains multipotent cells, including stem and stromal populations, of both the haematopoietic and skeletal systems. Bone marrow mesenchymal stromal cells (BMSCs) are characterised by tri-lineage differentiation (bone, cartilage and adipose tissue) and contribute to the formation of the bone marrow niche, which maintains haematopoietic stem cell quiescence. This quiescence ensures life-long haematopoiesis and the supply of mature blood cells to the haematopoietic system. In this study we investigate the interactions between haematopoietic and BMSCs (in both human and mouse cultures) specifically to understand the consequences on BMSCs during tissue repair.

A murine MSC cell-line model was co-cultured with enriched fractions of primary murine haematopoietic progenitor cells isolated based on c-Kit, Sca-1, and lineage markers. Similarly, human bone marrow derived MSCs were co-cultured with primary bone marrow haematopoietic fractions isolated based on CD34, CD38 and lineage markers. Using confocal microscopy, we demonstrated that the two cell populations directly interact through cell-cell contact with haematopoietic cells located above and below the MSC monolayer. Cultures were then pushed to differentiate down the osteogenic lineage. Results indicate that MSCs co-cultured with haematopoietic cells exhibited significant inhibition of osteogenesis when analysed by functional assay of matrix mineralisation and gene expression analysis for transcripts including Runx2, Osterix and type I collagen.

These data support the hypothesis that hematopoietic progenitor cells influence both the local homeostasis of the bone marrow as well as the repair potential of stromal cells. Such interactions could be important for the resolution of injury after trauma induced repair. Furthermore, manipulation of these interactions, such as the administration of haematopoietic cell stimulating agents, could be used to improve treatment outcomes.

Human mesenchymal stem cells (hMSCs) have the capacity to differentiate into adipocytes, chondrocytes, or osteoblasts, and are an exciting tool to be used in regenerative medicine and surgery. By manipulating the surface structure and physical properties of a biomaterial on which hMSCs can be incorporated, the biological response of these cells at the implant site can be controlled. Whilst both topography and surface stiffness are known to influence differentiation of hMSC's, little is understood of the molecular mechanisms that underpin these responses. In this study we use immunofluorescence and confocal microscopy techniques to assess the change in both the abundance and the distribution of H3K9me2 or H3K9ac patterns in hMSCs cultured on materials with controlled topography and stiffness, under basal and osteogenic conditions. These data demonstrate that levels and localisation of both H3K9me2 and H3K9ac alter in hMSCs cultured on the different substrates and that these surfaces dictate the response to osteogenic stimuli, suggesting that the control of cytoskeletal structure can be linked to chromatin activity. This regulation of histone modification by MSC interaction with the surrounding scaffold provides not only a mechanistic link to the control of cell fate but also the opportunity to design biomaterials that better influence cell activity.

hMSC cultures were prepared from osteoarthritic patients. Silicone elastomer (PDMS) culture surfaces of varying degrees of stiffness (1:10, 1:30 and 1:50 PDMS, tissue culture plastic and glass) were investigated in isolation and in combination with differentiation media. CD marker expressions of ‘stemness’ were investigated. RNA expression changes in OA-hMSCs and non-OA-hMSCs were also investigated for a panel of genes (inclusive of ‘stemness-’ and osteogenic-linked genes, FKBP5 and osteomodulin).

Degree of early integration of titanium alloy implants into bone is an important predictor of long term implant success in arthroplasty. The correlation between observations on early cell adhesion and the ability of modified surfaces to affect osseointegration of implants in in vivo models is unclear. We hypothesised that observation of increased focal adhesion complexes in early cultures of osteoblasts would correlate with increased osseointegration of treated implants in an animal model. Longer term culture of rat osteoblasts for alkaline phosphatase activity indicated that cells cultured on the 9V treated surfaces were displaying greater alkaline phosphatase activity at 14 days. Bone nodule formation at 28 days demonstrated a trend towards smaller area of bone nodules on the surfaces treated at 9V then those treated at 3V and 5V. A rat model was employed for testing mechanical push-out strength of experimental implants and demonstrated a trend towards increased yield strength of the bone-implant interface for implants treated at 3V180s and 5V180s. Histomorphometry was performed and no statistically significant differences in percentage area of contact with mineralised bone matrix were seen, although there was a trend for greater mineralised matrix contact on the polished and 9V180s treated implants. Previous experiments demonstrated cells on the 9V treated surfaces were well spread and had significantly increased size and number of focal adhesions. This was regarded as indicating more successful cell adhesion. The above results demonstrate that this early trend disappeared in longer term culture did not persist in experiments in an animal model.

Background

Uncemented implants are an important part of the arthroplasty armamentarium. Risk of aseptic loosening and failure of these components is related to initial osseointegration - the formation of a seamless bone-implant interface without interposition of fibrous tissue.

We used an atomic layer deposition (ALD) approach to create titanium oxide nanolayers on ultra high molecular weight polyethylene (UHMWPE) surfaces. These materials were then characterised in terms of rat osteoblast adhesion, morphology and differentiation.

UHMWPE discs produced from a machined cylinder or impact moulded discs were coated with titanium oxide by ALD. Light, atomic force microscopy and scanning electron microscopy with EDX were used to characterise the coated surfaces. These approaches showed 1-1.5 micron tooling grooves with a periodicity of 40 microns on the machined discs whilst the moulded discs exhibited nanotopographical features. The titanium oxide coating was successfully deposited on discs from both sources but was not uniform across the surfaces, with vein-like ‘creases’ clearly visible. We believe that these features are due to the thermal expansion of the UHMWPE discs during the ALD process and their subsequent cooling.

Coated and uncoated discs were seeded with osteoblasts for 24 hours, then fixed. Immunofluorescence microscopy and computer-based image processing enabled determination of osteoblast numbers, size and shape. A trend of larger average cell area was associated with the coated discs and P<0.01 for an H0 of no difference in cell area between coated and uncoated grooved discs.

Osteoblasts were also cultured on the discs in osteogenic medium to promote bone nodule formation. After a few weeks, von Kossa staining and computer-based image processing allowed calculation of surface area covered with bone nodules for each of the discs. Based on results from three of each type of disc, a significantly greater proportion of the surface area of coated discs was covered with calcified deposits compared to uncoated discs (P<0.025 for grooved discs and P<0.005 for smooth discs). On average, the coated discs had bone nodules on 1.4 times the surface area as compared to their uncoated counterparts.

The hypothesis for our study was that TiO2 coating of a polymer might better promote osteoblast interaction with the biomaterial surface leading to enhanced osteogenesis. Our preliminary data support this view and suggest that this approach could likely be exploited in the fabrication of implant materials with tailored biological activity.