Publications by authors named "Jehan J El-Jawhari"

23 Publications

  • Page 1 of 1

Bioengineering Approaches for Delivering Growth Factors: A Focus on Bone and Cartilage Regeneration.

Bioengineering (Basel) 2022 May 20;9(5). Epub 2022 May 20.

Biosciences Department, School of Science and Technology, Nottingham Trent University, Nottingham NG11 8PT, UK.

Growth factors are bio-factors that target reparatory cells during bone regeneration. These growth factors are needed in complicated conditions of bone and joint damage to enhance tissue repair. The delivery of these growth factors is key to ensuring the effectiveness of regenerative therapy. This review discusses the roles of various growth factors in bone and cartilage regeneration. The methods of delivery of natural or recombinant growth factors are reviewed. Different types of scaffolds, encapsulation, Layer-by-layer assembly, and hydrogels are tools for growth factor delivery. Considering the advantages and limitations of these methods is essential to developing regenerative therapies. Further research can accordingly be planned to have new or combined technologies serving this purpose.
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http://dx.doi.org/10.3390/bioengineering9050223DOI Listing
May 2022

Evaluation of Human Bone Marrow Mesenchymal Stromal Cell (MSC) Functions on a Biomorphic Rattan-Wood-Derived Scaffold: A Comparison between Cultured and Uncultured MSCs.

Bioengineering (Basel) 2021 Dec 21;9(1). Epub 2021 Dec 21.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds LS9 7TF, UK.

The reconstruction of large bone defects requires the use of biocompatible osteoconductive scaffolds. These scaffolds are often loaded with the patient's own bone marrow (BM) cells to facilitate osteoinductivity and biological potency. Scaffolds that are naturally sourced and fabricated through biomorphic transitions of rattan wood (B-HA scaffolds) offer a unique advantage of higher mechanical strength and bioactivity. In this study, we investigated the ability of a biomorphic B-HA scaffold (B-HA) to support the attachment, survival and gene expression profile of human uncultured BM-derived mesenchymal stromal cells (BMSCs, = 6) and culture expanded MSCs (cMSCs, = 7) in comparison to a sintered, porous HA scaffold (S-HA). B-HA scaffolds supported BMSC attachment (average 98%) and their survival up to 4 weeks in culture. Flow cytometry confirmed the phenotype of cMSCs on the scaffolds. Gene expression indicated clear segregation between cMSCs and BMSCs with MSC osteogenesis- and adipogenesis-related genes including and being higher expressed in BMSCs. These data indicated a unique transcriptional signature of BMSCs that was distinct from that of cMSCs regardless of the type of scaffold or time in culture. There was no statistical difference in the expression of osteogenic genes in BMSCs or cMSCs in B-HA compared to S-HA. VEGF release from cMSCs co-cultured with human endothelial cells ( = 4) on B-HA scaffolds suggested significantly higher supernatant concentration with endothelial cells on day 14. This indicated a potential mechanism for providing vasculature to the repair area when such scaffolds are used for treating large bone defects.
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http://dx.doi.org/10.3390/bioengineering9010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8773042PMC
December 2021

Bone Marrow Multipotent Mesenchymal Stromal Cells as Autologous Therapy for Osteonecrosis: Effects of Age and Underlying Causes.

Bioengineering (Basel) 2021 May 17;8(5). Epub 2021 May 17.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, UK.

Bone marrow (BM) is a reliable source of multipotent mesenchymal stromal cells (MSCs), which have been successfully used for treating osteonecrosis. Considering the functional advantages of BM-MSCs as bone and cartilage reparatory cells and supporting angiogenesis, several donor-related factors are also essential to consider when autologous BM-MSCs are used for such regenerative therapies. Aging is one of several factors contributing to the donor-related variability and found to be associated with a reduction of BM-MSC numbers. However, even within the same age group, other factors affecting MSC quantity and function remain incompletely understood. For patients with osteonecrosis, several underlying factors have been linked to the decrease of the proliferation of BM-MSCs as well as the impairment of their differentiation, migration, angiogenesis-support and immunoregulatory functions. This review discusses the quality and quantity of BM-MSCs in relation to the etiological conditions of osteonecrosis such as sickle cell disease, Gaucher disease, alcohol, corticosteroids, Systemic Lupus Erythematosus, diabetes, chronic renal disease and chemotherapy. A clear understanding of the regenerative potential of BM-MSCs is essential to optimize the cellular therapy of osteonecrosis and other bone damage conditions.
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http://dx.doi.org/10.3390/bioengineering8050069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156020PMC
May 2021

Interleukin-7: a potential factor supporting B-cell maturation in the rheumatoid arthritis synovium.

Clin Exp Rheumatol 2021 Mar-Apr;39(2):253-262. Epub 2021 Mar 21.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, and NIHR Leeds Musculoskeletal Biomedical Research Centre, Chapel Allerton Hospital, Leeds, UK.

Objectives: The exact function of interleukin-7 (IL-7) in autoimmune diseases remains unclear although it is a recognised therapeutic target for cytokine blockade. Our objective was to investigate the regulation and downstream effect of IL-7 in diseased tissue from rheumatoid arthritis (RA) patients notably with respect to its function as bone turnover regulator and tissue architecture (TA) organiser.

Methods: Synovial tissues (fresh, frozen or xed) were obtained from our tissue bank and distributed between experiments for live cell cultures, histology, immunohistochemistry or gene expression array by qPCR.

Results: IL-7 expression in synoviocyte cultures was up-regulated by pro-in ammatory cytokines, notably IL-6. Gene expression pro ling segregated synovial biopsies based on the presence of B/plasma cells and ectopic TA. IL-7 gene expression was associated with that of several genes whose function was to support B-cell maturation in tissue with distinct B-cell aggregates (despite the lack of IL-7-Receptor expression on B-cells) as well as with ectopic germinal-like centres. IL-7 was associated with bone turnover regulation in biopsies with diffuse in ltration. A novel relationship between the IL-7 and IL-6 axis was also highlighted in human tissue.

Conclusions: Overall, IL-7 may contribute to the maintenance of the pro-in ammatory cycle perpetuating in ammation in RA synovium. We therefore propose a novel role for IL-7 as an orchestrator of TA with an impact on B-cell maturation in relation with IL-6.
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April 2021

Multipotent Mesenchymal Stromal Cells in Rheumatoid Arthritis and Systemic Lupus Erythematosus; From a Leading Role in Pathogenesis to Potential Therapeutic Saviors?

Front Immunol 2021 24;12:643170. Epub 2021 Feb 24.

Faculty of Medicine and Health, Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.

The pathogenesis of the autoimmune rheumatological diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is complex with the involvement of several immune cell populations spanning both innate and adaptive immunity including different T-lymphocyte subsets and monocyte/macrophage lineage cells. Despite therapeutic advances in RA and SLE, some patients have persistent and stubbornly refractory disease. Herein, we discuss stromal cells' dual role, including multipotent mesenchymal stromal cells (MSCs) also used to be known as mesenchymal stem cells as potential protagonists in RA and SLE pathology and as potential therapeutic vehicles. Joint MSCs from different niches may exhibit prominent pro-inflammatory effects in experimental RA models directly contributing to cartilage damage. These stromal cells may also be key regulators of the immune system in SLE. Despite these pro-inflammatory roles, MSCs may be immunomodulatory and have potential therapeutic value to modulate immune responses favorably in these autoimmune conditions. In this review, the complex role and interactions between MSCs and the haematopoietically derived immune cells in RA and SLE are discussed. The harnessing of MSC immunomodulatory effects by contact-dependent and independent mechanisms, including MSC secretome and extracellular vesicles, is discussed in relation to RA and SLE considering the stromal immune microenvironment in the diseased joints. Data from translational studies employing MSC infusion therapy against inflammation in other settings are contextualized relative to the rheumatological setting. Although safety and proof of concept studies exist in RA and SLE supporting experimental and laboratory data, robust phase 3 clinical trial data in therapy-resistant RA and SLE is still lacking.
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http://dx.doi.org/10.3389/fimmu.2021.643170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959804PMC
September 2021

Multipotential stromal cells in the talus and distal tibia in ankle osteoarthritis - Presence, potency and relationships to subchondral bone changes.

J Cell Mol Med 2021 01 11;25(1):259-271. Epub 2020 Dec 11.

Faculty of Medicine and Health, Leeds Institute of Rheumatoid and Musculoskeletal Medicine, University of Leeds, Leeds, UK.

A large proportion of ankle osteoarthritis (OA) has an early onset and is post-traumatic. Surgical interventions have low patient satisfaction and relatively poor clinical outcome, whereas joint-preserving treatments, which rely on endogenous multipotential stromal cells (MSCs), result in suboptimal repair. This study investigates MSC presence and potency in OA-affected talocrural osteochondral tissue. Bone volume fraction (BV/TV) changes for the loading region trabecular volume and subchondral bone plate (SBP) thickness in OA compared with healthy tissue were investigated using microcomputed tomography. CD271-positive MSC topography was related to bone and cartilage damage in OA tissue, and in vitro MSC potency was compared with control healthy iliac crest (IC) MSCs. A 1.3- to 2.5-fold SBP thickening was found in both OA talus and tibia, whereas BV/TV changes were depth-dependent. MSCs were abundant in OA talus and tibia, with similar colony characteristics. Tibial and talar MSCs were tripotential, but talar MSCs had 10-fold lower adipogenesis and twofold higher chondrogenesis than IC MSCs (P = .01 for both). Cartilage damage in both OA tibia and talus correlated with SBP thickening and CD271+ MSCs was 1.4- to twofold more concentrated near the SBP. This work shows multipotential MSCs are present in OA talocrural subchondral bone, with their topography suggesting ongoing involvement in SBP thickening. Potentially, biomechanical stimulation could augment the chondrogenic differentiation of MSCs for joint-preserving treatments.
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http://dx.doi.org/10.1111/jcmm.15993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810934PMC
January 2021

Receptor activator of nuclear factor kappa-Β ligand (RANKL) serum levels are associated with progression to seropositive/negative rheumatoid arthritis.

Clin Exp Rheumatol 2021 May-Jun;39(3):456-462. Epub 2020 Jul 21.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, and Leeds Teaching Hospitals NHS Trust, Chapel Allerton Hospital, Leeds, UK.

Objectives: The aim of this study was to establish whether serum RANKL levels in early inflammatory arthritis (IA) were associated with rheumatoid arthritis (RA) diagnosis at follow-up, and to evaluate the added value of RANKL for RA diagnosis.

Methods: Serum from 298 patients was collected. Demographic and clinical (swollen/tender joint counts, CRP, DAS28-CRP, RF, ACPA and shared-epitope data were recorded. Baseline ultrasound of 26 joints was performed, including total power Doppler (PD). An ELISA was used to measure RANKL. Predictors of progression were identified using multivariable logistic regression analysis. Area under the receiver operating characteristics (AUROC) was used to assess the performance of the prediction models and quantify the added value of RANKL in RA diagnosis.

Results: 151 patients developed RA and 147 were non-RA (undifferentiated IA, other inflammatory diagnoses or non-persistent inflammation). RANKL levels were significantly higher in RA (median [IQR]: 474.1 [270.8-1430.6]) than in non-RA (median [IQR]: 301.0 [174.1-477.5]. Three clinical factors (age, SJC and PD) were identified by multivariable logistic regression with model performance AUROC of 77.9% (95% CI 72.1-83.8%). Adding RANKL resulted in a relative increase of 6.5% in the model classification performance of an AUROC of 83.0% (95% CI 77.9-88.1%). In ACPA-negative patients, the model performance increased from 77.6% (95% CI 69.5-85.7%) with clinical data only to 81.9% (95% CI 73.7-89.8%) with added value of RANKL and imaging.

Conclusions: RANKL levels can predict RA diagnosis over clinical biomarkers alone, both seropositive and particularly in seronegative IA patients.
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May 2021

Intrinsic Type 1 Interferon (IFN1) Profile of Uncultured Human Bone Marrow CD45CD271 Multipotential Stromal Cells (BM-MSCs): The Impact of Donor Age, Culture Expansion and IFNα and IFNβ Stimulation.

Biomedicines 2020 Jul 15;8(7). Epub 2020 Jul 15.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds LS9 7TF, UK.

Skeletal aging is associated with reduced proliferative potential of bone marrow (BM) multipotential stromal cells (MSCs). Recent data suggest the involvement of type 1 interferon (IFN1) signalling in hematopoietic stem cell (HSC) senescence. Considering that BM-HSCs and BM-MSCs share the same BM niche, we investigated IFN1 expression profile in human BM-MSCs in relation to donor age, culture-expansion and IFN1 (α and β) stimulation. Fluorescence-activated cell sorting was used to purify uncultured BM-MSCs from younger (19-41, = 6) and older (59-89, = 6) donors based on the CD45CD271 phenotype, and hematopoietic-lineage cells (BM-HLCs, CD45CD271) were used as controls. Gene expression was analysed using integrated circuits arrays in sorted fractions as well as cultured/stimulated BM-MSCs and Y201/Y202 immortalised cell lines. IFN1 stimulation led to BM-MSC growth arrest and upregulation of many IFN1-stimulated genes (ISGs), with IFNβ demonstrating stronger effects. Uncultured MSCs were characterised by a moderate-level ISG expression similar to Y201 cells. Age-related changes in ISG expression were negligible in BM-MSCs compared to BM-HLCs, and intracellular reactive oxygen species (ROS) levels in BM-MSCs did not significantly correlate with donor age. Antiaging genes Klotho and SIRT6 correlated with more ISGs in BM-MSCs than in BM-HLCs. In patients with osteoarthritis (OA), BM-MSCs expressed considerably lower levels of several ISGs, indicating that their IFN1 signature is affected in a pathological condition. In summary, BM-MSCs possess homeostatic IFN1 gene expression signature in health, which is sensitive to in vitro culture and external IFN1 stimulation. IFN signalling may facilitate in vivo BM-MSC responses to DNA damage and combating senescence and aberrant immune activation.
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http://dx.doi.org/10.3390/biomedicines8070214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399891PMC
July 2020

The Biological Fitness of Bone Progenitor Cells in Reamer/Irrigator/Aspirator Waste.

J Bone Joint Surg Am 2019 Dec;101(23):2111-2119

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.

Background: The biological waste collected during use of the Reamer/Irrigator/Aspirator (RIA; DePuy Synthes) has been described as an abundant source of bone progenitor cells with a comparable osteogenic gene profile to donor-matched iliac crest bone marrow (IC-BM). However, it is not clear whether these RIA-waste (RIA-W) cells are biologically fit. We aimed to evaluate the stress levels and functions of RIA-W progenitor cells.

Methods: Reactive oxygen species (ROS) levels were tested in freshly collected bone progenitor cells (defined as CD45CD271 cells) using flow cytometry. ROS levels induced in these cells by hypoxia and/or oxidative stress as well as by an experimental simulation of the RIA procedure were also measured. Furthermore, the alkaline phosphatase (ALP) expression levels, proliferation, and senescence of culture-expanded RIA-W and IC-BM mesenchymal stromal cells (MSCs) were compared.

Results: RIA-W and donor-matched IC-BM CD45CD271 cells were 97% and 98% viable, but the ROS levels were significantly higher for RIA-W cells than for IC-BM cells (p = 0.0020). Also, ROS induced by hypoxia, oxidative stress, and both were higher for RIA-W cells (p = 0.0312, 0.0156, and 0.0703, respectively). Dilution with saline solution, suction pressure, and irrigation reduced cell viability, with a positive correlation with the ROS level (p = 0.0035). The RIA-W and IC-BM colony-forming cells (average, 96,100 and 11,500, respectively) showed comparable ALP levels. Furthermore, culture-expanded RIA-W and IC-BM MSCs showed comparable ROS levels, ALP levels, susceptibility to death, and proliferation.

Conclusions: Although freshly collected RIA-W bone progenitor cells appeared to be transiently stressed, these cells were as viable as IC-BM cells and present in greater numbers. The proliferation and osteogenesis of both cell types were comparable.

Clinical Relevance: The RIA waste bag contains bone progenitor cells with promising potential for regenerative applications, and should not be wasted.
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http://dx.doi.org/10.2106/JBJS.19.00133DOI Listing
December 2019

Defective Proliferation and Osteogenic Potential with Altered Immunoregulatory phenotype of Native Bone marrow-Multipotential Stromal Cells in Atrophic Fracture Non-Union.

Sci Rep 2019 11 22;9(1):17340. Epub 2019 Nov 22.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, School of Medicine, University of Leeds, Leeds, UK.

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271CD45 cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271CD45 cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing.
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http://dx.doi.org/10.1038/s41598-019-53927-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874596PMC
November 2019

The Analysis of Aging in Human Bone Marrow Mesenchymal Stromal Cells Using Colony-Forming Unit-Fibroblast Assay and the CD45CD271 Phenotype.

Stem Cells Int 2019 1;2019:5197983. Epub 2019 Aug 1.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK.

Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45CD271 cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay ( = -0.527, < 0.0001) or flow cytometry ( = -0.307, = 0.0116). Compared to young donors (19-40 years old), colony IDs were significantly lower in older donors (61-89 years old), particularly for smaller-sized colonies (42% lower, < 0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, < 0.0001, and 27%, < 0.05, respectively, indicating the formation of smaller sparser colonies. No significant differences in the expression of selected adipogenic, osteogenic, stromal, and bone remodelling genes as well as CD295, CD146, CD106, and connexin 43 surface molecules were found in sorted CD45CD271 MSCs from young and old donors ( = 8 donors each). Altogether, these results show similar trends for age-related decline in BM MSC numbers measured by the CFU-F assay and flow cytometry and reveal age-related effects of human serum on MSC colony formation. No significant differences in selected gene expression in uncultured CD45CD271 MSCs suggest that old donor MSCs may not be inferior in regard to their multipotential functions. Due to large donor-to-donor variation in all donor groups, our data indicate that an individual's chronological age is not a reliable predictor of their MSC number or potency.
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http://dx.doi.org/10.1155/2019/5197983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701348PMC
August 2019

Gene expression and functional comparison between multipotential stromal cells from lateral and medial condyles of knee osteoarthritis patients.

Sci Rep 2019 06 27;9(1):9321. Epub 2019 Jun 27.

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.

Osteoarthritis (OA) is the most common degenerative joint disorder. Multipotential stromal cells (MSCs) have a crucial role in joint repair, but how OA severity affects their characteristics remains unknown. Knee OA provides a good model to study this, as osteochondral damage is commonly more severe in the medial weight-bearing compartment compared to lateral side of the joint. This study utilised in vitro functional assays, cell sorting, gene expression and immunohistochemistry to compare MSCs from medial and lateral OA femoral condyles. Despite greater cartilage loss and bone sclerosis in medial condyles, there was no significant differences in MSC numbers, growth rates or surface phenotype. Culture-expanded and freshly-purified medial-condyle MSCs expressed higher levels of several ossification-related genes. Using CD271-staining to identify MSCs, their presence and co-localisation with TRAP-positive chondroclasts was noted in the vascular channels breaching the osteochondral junction in lateral condyles. In medial condyles, MSCs were additionally found in small cavities within the sclerotic plate. These data indicate subchondral MSCs may be involved in OA progression by participating in cartilage destruction, calcification and sclerotic plate formation and that they remain abundant in severe disease. Biological or biomechanical modulation of these MSCs may be a new strategy towards cartilage and bone restoration in knee OA.
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http://dx.doi.org/10.1038/s41598-019-45820-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597541PMC
June 2019

Optimising proliferation and migration of mesenchymal stem cells using platelet products: A rational approach to bone regeneration.

J Orthop Res 2019 06 28;37(6):1329-1338. Epub 2019 Mar 28.

Academic Department of Trauma and Orthopaedic Surgery, Leeds General Infirmary, Leeds, England.

This study investigates how mesenchymal stem cell's (MSCs) proliferation and migration abilities are influenced by various platelet products (PP). Donor-matched, clinical-, and control laboratory-standard PPs were generated and assessed based on their platelet and leukocyte concentrations. Bone marrow derived MSCs were exposed to these PP to quantify their effect on in vitro MSC proliferation and migration. An adapted colony forming unit fibroblast (CFU-F) assay was carried out on bone marrow aspirate using clinical-standard PP-loaded electrospun poly(ϵ-caprolactone) (PCL) membrane to mimic future clinical applications to contain bone defects. Clinical-standard PP had lower platelet (2.5 fold, p < 0.0001) and higher leukocyte (14.1 fold, p < 0.0001) concentrations compared to laboratory-standard PP. It induced suboptimal MSC proliferation compared to laboratory-standard PP and fetal calf serum (FCS). All PP induced significantly more MSC migration than FCS up to 24 h. The removal of leukocytes from PP had no effect on MSC proliferation or migration. The PP-loaded membranes successfully supported MSC colony formation. This study indicates that platelet concentrations in PP impact MSC proliferation more than the presence of leukocytes, whilst MSC migration in response to PP is not influenced by platelet or leukocyte numbers. Clinical-standard PP could be applied alongside manufactured membranes in the future treatment of bone reconstruction. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1329-1338, 2019.
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http://dx.doi.org/10.1002/jor.24261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065095PMC
June 2019

T cell immunomodulation by clinically used allogeneic human cancellous bone fragments: a potential novel immunotherapy tool.

Sci Rep 2018 09 10;8(1):13535. Epub 2018 Sep 10.

National Institute of Health Research Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK.

Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.
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http://dx.doi.org/10.1038/s41598-018-31979-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131386PMC
September 2018

The regenerative therapies of the ankle degeneration: a focus on multipotential mesenchymal stromal cells.

Regen Med 2018 Mar 19. Epub 2018 Mar 19.

Leeds Institute of Rheumatic & Musculoskeletal Medicine, University of Leeds, Leeds, UK.

The ankle degeneration ranging from focal osteochondral lesions to osteoarthritis can cause a total joint function loss. With rising life expectancy and activity of the patients, various regenerative therapies were introduced aiming to preserve the joint function via the induction of cartilage and bone repair. Here, biological events and mechanical changes of the ankle degeneration were discussed. The regenerative therapies were reviewed versus the standard surgical treatment. We especially focused on the use of mesenchymal (multipotential) stromal cells (MSCs) highlighting their dual functions of regeneration and cell modulation with an emphasis on the emerging MSC-based clinical studies. Being at an early step, more basic and clinical research is needed to optimize the applications of all ankle regenerative therapies including MSC-based methods.
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http://dx.doi.org/10.2217/rme-2017-0104DOI Listing
March 2018

Age-related Changes in Bone Marrow Mesenchymal Stromal Cells: A Potential Impact on Osteoporosis and Osteoarthritis Development.

Cell Transplant 2017 09;26(9):1520-1529

1 Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.

Aging at the cellular level is a complex process resulting from accumulation of various damages leading to functional impairment and a reduced quality of life at the level of the organism. With a rise in the elderly population, the worldwide incidence of osteoporosis (OP) and osteoarthritis (OA) has increased in the past few decades. A decline in the number and "fitness" of mesenchymal stromal cells (MSCs) in the bone marrow (BM) niche has been suggested as one of the factors contributing to bone abnormalities in OP and OA. It is well recognized that MSCs in vitro acquire culture-induced aging features such as gradual telomere shortening, increased numbers of senescent cells, and reduced resistance to oxidative stress as a result of serial population doublings. In contrast, there is only limited evidence that human BM-MSCs "age" similarly in vivo. This review compares the various aspects of in vitro and in vivo MSC aging and suggests how our current knowledge on rejuvenating cultured MSCs could be applied to develop future strategies to target altered bone formation processes in OP and OA.
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http://dx.doi.org/10.1177/0963689717721201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680949PMC
September 2017

The CD45lowCD271high Cell Prevalence in Bone Marrow Samples May Provide a Useful Measurement of the Bone Marrow Quality for Cartilage and Bone Regenerative Therapy.

J Bone Joint Surg Am 2017 Aug;99(15):1305-1313

1Leeds Institute of Rheumatic and Musculoskeletal Medicine, St. James Hospital, University of Leeds, Leeds, United Kingdom 2NIHR-Leeds Musculoskeletal Biomedical Research Unit (LMBRU), Chapel Allerton Hospital, University of Leeds, Leeds, United Kingdom 3Clinical Pathology Department, Mansoura University, Mansoura, Egypt.

Background: Bone marrow aspirates and concentrates are increasingly being used for musculoskeletal regenerative therapies, providing bone and cartilage progenitors. However, the quality of these bone marrow samples remains imprecise within clinical settings. As there is a need for the development of these therapies, a method of counting CD45CD271 cells was optimized and tested as an indicator of bone marrow sample quality.

Methods: Bone marrow aspirates were collected from 54 donors (28 male and 26 female; median age of 48 years). The reagent concentrations were optimized for fast staining, and an acoustic-focusing flow cytometer (Attune) was used to enable automated CD45CD271 cell counting in bone marrow aspirates, bone marrow concentrates, and samples loaded onto a collagen scaffold. The CD45CD271 cell counts were compared with those obtained using another flow-cytometry-based method (LSR II) and with connective tissue progenitor (CTP) counts quantified using a colony forming unit-fibroblast (CFU-F) assay.

Results: The optimized method enabled the counting of CD45CD271 cells within only 15 minutes. The quantified cell counts (median, 1,520; range, 96 to 20,992 cells/mL of bone marrow) were positively correlated with the CTP counts (p < 0.0001; r = 0.7237). In agreement with CFU-F and LSR II-based assays, the CD45CD271 cell counts quantified using the Attune-based method decreased with age in the samples from female but not male donors (p = 0.0015 and p = 0.3877, respectively). A significant increase in CD45CD271 cell counts was detected following bone marrow concentration (mean, 5-fold; 95% confidence interval [CI], 3.6 to 7.2-fold). Additionally, the number of CD45CD271 cells attached to the collagen scaffold was positively correlated with the number of progenitor cells that survived on the scaffold after 2-week culture (p = 0.0348).

Conclusions: An assay for counting CD45CD271 cells may provide a useful measurement of bone marrow quality. While the specificity of this measurement of CD45CD271 cells remained low in our experimental conditions, CD45CD271 cell counts were positively and modestly correlated with the prevalence of CTPs.

Clinical Relevance: A fast and automated assessment of bone marrow aspirate/concentrate quality using CD45CD271 cell counting may be a useful tool for improving the quality of regenerative therapy.
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http://dx.doi.org/10.2106/JBJS.16.01138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125756PMC
August 2017

Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone formation in spondyloarthropathies.

Rheumatology (Oxford) 2017 Mar;56(3):488-493

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK.

Objectives.: The SpAs are genetically and therapeutically linked to IL-23, which in turn regulates IL-22, a cytokine that has been implicated in the regulation of new bone formation in experimental models. We hypothesize that IL-22, a master regulator of stem cells in other niches, might also regulate human mesenchymal stem cell (MSC) osteogenesis.

Methods.: The effects of IL-22 on in vitro MSC proliferation, migration and osteogenic differentiation were evaluated in the presence or absence of IFN-γ and TNF (to ascertain IL-22 activity in pro-inflammatory environments). Colorimetric XTT assay, trans-well migration assays, quantitative real-time PCR (qRT-PCR) for MSC lineage markers and osteogenesis assays were used.

Results.: Combined treatment of MSC with IL-22, IFN-γ and TNF resulted in increased MSC proliferation ( P = 0.008) and migration ( P = 0.04), an effect that was not seen in cells treated with IL-22 alone and untreated cells. Osteogenic and adipogenic, but not chondrogenic, transcription factors were upregulated by IL-22 alone ( P < 0.05). MSC osteogenesis was enhanced following IL-22 exposure ( P = 0.03, measured by calcium production). The combination of IFN-γ and TNF with or without IL-22 suppressed MSC osteogenesis ( P = 0.03).

Conclusion.: This work shows that IL-22 is involved in human MSC proliferation/migration in inflammatory environments, with MSC osteogenesis occurring only in the absence of IFN-γ/TNF. These effects of IL-22 on MSC function is a novel pathway for exploring pathological, post-inflammation osteogenesis in human SpA.
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http://dx.doi.org/10.1093/rheumatology/kew384DOI Listing
March 2017

The roles of immune cells in bone healing; what we know, do not know and future perspectives.

Injury 2016 Nov;47(11):2399-2406

Leeds Institute of Rheumatic and Musculoskeletal Medicine, St. James Hospital, University of Leeds, UK; NIHR Biomedical Research Unit, Chapel Allerton Hospital, University of Leeds, UK. Electronic address:

Key events occurring during the bone healing include well-orchestrated and complex interactions between immune cells, multipotential stromal cells (MSCs), osteoblasts and osteoclasts. Through three overlapping phases of this physiological process, innate and adaptive immune cells, cytokines and chemokines have a significant role to play. The aim of the escalating immune response is to achieve an osseous healing in the shortest time and with the least complications facilitating the restoration of function. The uninterrupted progression of these biological events in conjunction with a favourable mechanical environment (stable fracture fixation) remains the hallmark of successful fracture healing. When failure occurs, either the biological environment or the mechanical one could have been disrupted. Not infrequently both may be compromised. Consequently, regenerative treatments involving the use of bone autograft, allograft or synthetic matrices supplemented with MSCs are increasingly used. A better understanding of the bone biology and osteoimmunology can help to improve these evolving cell-therapy based strategies. Herein, an up to date status of the role of immune cells during the different phases of bone healing is presented. Additionally, the known and yet to know events about immune cell interactions with MSCs and osteoblasts and osteoclasts and the therapeutic implications are being discussed.
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http://dx.doi.org/10.1016/j.injury.2016.10.008DOI Listing
November 2016

Collagen-containing scaffolds enhance attachment and proliferation of non-cultured bone marrow multipotential stromal cells.

J Orthop Res 2016 Apr 1;34(4):597-606. Epub 2015 Dec 1.

Academic Unit of Musculoskeletal Disease, Leeds Institute of Rheumatic and Musculoskeletal Medicine, St. James University Hospital, University of Leeds, LS9 7TF, Leeds, United Kingdom.

Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen-containing bovine bone scaffold (Orthoss(®) Collagen) with a non-collagen-containing bovine bone scaffold, Orthoss(®) . Another collagen-containing synthetic scaffold, Vitoss(®) was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit-fibroblast assay and flow-cytometry. The number of BM MSCs initially attached to Orthoss(®) Collagen and Vitoss(®) was similar but greater than Orthoss(®) (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss(®) Collagen and Vitoss(®) after 2-week culture was also higher compared to Orthoss(®) (p = 0.010 and p = 0.023, respectively). Interestingly, collagen-containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture-expanded MSCs on Orthoss(®) collagen and Vitoss(®) was greater compared to Orthoss(®) (p = 0.047 and p = 0.004, respectively). Collectively, collagen-containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes.
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http://dx.doi.org/10.1002/jor.23070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063164PMC
April 2016

Modulation of peripheral T-cell function by interleukin-7 in rheumatoid arthritis.

Arthritis Res Ther 2014 Dec 23;16(6):511. Epub 2014 Dec 23.

Introduction: Interleukin-7 (IL-7) is a cytokine essential for T-cell lymphopoiesis, survival and polarization with an emerging role in autoimmunity. We previously demonstrated reduced levels of circulating IL-7 in rheumatoid arthritis (RA), although high amounts are expressed in joints, suggesting differences between systemic and synovial effects. We observed healthy levels of IL-7 in 48% of RA patients in clinical remission (CR) and aimed to investigate the consequences of IL-7 deficiency on T-cell responses.

Methods: We used RA patients with active disease and in CR presenting various levels of IL-7, to investigate its modulatory effects on T cells by analysing responses to phyto-haemagglutinin (PHA), expression of polarization or survival factors, or suppression by regulatory T cells (Tregs).

Results: IL-7 levels were normal (>10 pg/ml) in 48% of RA patients in CR. Amongst 63 CR patients followed up for 18 months, lack of IL-7 recovery was observed in 13 out of 15 (86%) patients experiencing relapse but only 11 out of 48 (23%) of those who did not (P = 0.0002). Binary regressions showed high significance for below normal IL-7 levels for self-reported maternal family history of arthritis (odds ratio (OR): 7.66, P = 0.006) and a trend for smoking (OR: 3.33, P = 0.068) with no further demographic or clinical associations. Serum IL-7 correlated with restored CD4+T-cell response to PHA (rho = 0.879); this was not related to an increase in T-cell proliferation capacity or expression of survival factors B-cell lymphoma 2 (BCL2) and BCL2-associated protein X (BAX). Expression of Th1 polarization factor (TBET) was also dependent on exposure to IL-7 in vivo (rho = 0.600). In contrast CD25highTregs' response to PHA was not affected by in vivo IL-7, but their suppression capabilities were related to circulating IL-7 (rho = 0.589). Co-stimulation with IL-7 (mimicking the joint environment) increased responsiveness of CD4+T-cells to PHA, lowering the ability of CD25highTregs to suppress them.

Conclusions: Our data demonstrate that IL-7 has a critical role in modulating T-cell function in vivo, possibly explaining opposing effects observed systemically and in the joint. Lack of IL-7 recovery in CR by maintaining a suppressed immune system may be a determinant factor in the occurrence of relapse.
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http://dx.doi.org/10.1186/s13075-014-0511-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298067PMC
December 2014

Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes.

Mol Immunol 2014 Apr 22;58(2):160-8. Epub 2013 Dec 22.

Leeds Institute of Molecular Medicine, University of Leeds, Wellcome Brenner Building, St. James's University Hospital, Leeds LS9 7TF, UK. Electronic address:

Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.
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http://dx.doi.org/10.1016/j.molimm.2013.11.020DOI Listing
April 2014

Human tumour immune evasion via TGF-β blocks NK cell activation but not survival allowing therapeutic restoration of anti-tumour activity.

PLoS One 2011 6;6(9):e22842. Epub 2011 Sep 6.

Leeds Institute of Molecular Medicine, Wellcome Brenner Building, St. James's University Hospital, University of Leeds, Leeds, United Kingdom.

Immune evasion is now recognized as a key feature of cancer progression. In animal models, the activity of cytotoxic lymphocytes is suppressed in the tumour microenvironment by the immunosuppressive cytokine, Transforming Growth Factor (TGF)-β. Release from TGF-β-mediated inhibition restores anti-tumour immunity, suggesting a therapeutic strategy for human cancer. We demonstrate that human natural killer (NK) cells are inhibited in a TGF-β dependent manner following chronic contact-dependent interactions with tumour cells in vitro. In vivo, NK cell inhibition was localised to the human tumour microenvironment and primary ovarian tumours conferred TGF-β dependent inhibition upon autologous NK cells ex vivo. TGF-β antagonized the interleukin (IL)-15 induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of both NK cell activation receptor molecules and components of the cytotoxic apparatus. Interleukin-15 also promotes NK cell survival and IL-15 excluded the pro-apoptotic transcription factor FOXO3 from the nucleus. However, this IL-15 mediated pathway was unaffected by TGF-β treatment, allowing NK cell survival. This suggested that NK cells in the tumour microenvironment might have their activity restored by TGF-β blockade and both anti-TGF-β antibodies and a small molecule inhibitor of TGF-β signalling restored the effector function of NK cells inhibited by autologous tumour cells. Thus, TGF-β blunts NK cell activation within the human tumour microenvironment but this evasion mechanism can be therapeutically targeted, boosting anti-tumour immunity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022842PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167809PMC
January 2012
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