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It is well known that the biomechanical and tribological performance of articular cartilage is inextricably linked to its extracellular matrix (ECM) structure and zonal heterogeneity. Furthermore, it is understood that the presence of native ECM components, such as collagen II and aggrecan, promote healthy homeostasis in the resident chondrocytes. What is less frequently discussed is how chondrocyte metabolism is related to the extracellular mechanical environment, at both the macro and microscale. The chondrocyte is in immediate contact with the pericellular matrix of the chondron, which acts as a mechanocoupler, transmitting external applied loads from the ECM to the chondrocyte. Therefore, components of the pericellular matrix also play essential roles in chondrocyte mechanotransduction and metabolism. Recreating the biomechanical environment through tuning material properties of a scaffold and/or the use of external cyclic loading can induce biosynthetic responses in chondrocytes. Decellularized scaffolds, which retain the native tissue macro- and microstructure also represent an effective means of recapitulating such an environment. The use of such techniques in tissue engineering applications can ensure the regeneration of skeletally mature articular cartilage with appropriate biomechanical and tribological properties to restore joint function. Despite the pivotal role in graft maturation and performance, biomechanical and tribological properties of such interventions is often underrepresented. This review outlines the role of biomechanics in relation to native cartilage performance and chondrocyte metabolism, and how application of this theory can enhance the future development and successful translation of biomechanically relevant tissue engineering interventions. Impact statement Physiological cartilage function is a key criterion in the success of a cartilage tissue engineering solution. The

Platelet products (PP) and bone-marrow aspirate are popular sources of osteoinductive signalling molecules and osteogenic bone marrow mesenchymal stromal cells (BM-MSCs) used in the treatment of impaired bone healing. However, the combined use of PP and BM-MSCs in clinical studies has reported mixed results. Understanding the cellular and molecular interactions between PP and BM-MSCs plays the important role of guiding future research and clinical application. This systematic review investigates the effects of PP on the biophysiological functions of BM-MSCs in in vitro human studies, including (i) proliferation, (ii) migration, (iii) differentiation, (iv) growth factor/cytokine/protein expression, (v) immunomodulation, (vi) chemotactic effect on haematopoietic stem cells, (vii) response to apoptotic stress, and (viii) gene expression. In vitro studies in human have demonstrated the multi-faceted ‘priming effect’ of PP on the biophysiological functions of BM-MSCs. PP has been shown to improve proliferation, migration, osteogenic differentiation, reaction to apoptotic stress as well as immunomodulatory, pro-angiogenic and pro-inflammatory capacities of BM-MSCs. Several factors are highlighted that restrict the transferability of these findings into clinical practice. Therefore, more collaborative in vitro research in humans modelled to reflect clinical practice is required to better understand the effects of PP exposure on the biophysiological function(s) of BM-MSCs in human.

A curious feature of axial disease in ankylosing spondylitis (AS) and related non-radiographic axial spondyloarthropathy (nrAxSpA) is that spinal inflammation may ultimately be associated with excessive entheseal tissue repair with new bone formation. Other SpA associated target tissues including the gut and the skin have well established paradigms on how local tissue immune responses and proven disease relevant cytokines including TNF and the IL-23/17 axis contribute to tissue repair. Normal skeletal homeostasis including the highly mechanically stressed entheseal sites is subject to tissue microdamage, micro-inflammation and ultimately repair. Like the skin and gut, healthy enthesis has resident immune cells including ILCs, γδ T cells, conventional CD4+ and CD8+ T cells and myeloid lineage cells capable of cytokine induction involving prostaglandins, growth factors and cytokines including TNF and IL-17 that regulate these responses. We discuss how human genetic studies, animal models and translational human immunology around TNF and IL-17 suggest a largely redundant role for these pathways in physiological tissue repair and homeostasis. However, disease associated immune system overactivity of these cytokines with loss of tissue repair “fine tuning” is eventually associated with exuberant tissue repair responses in AS. Conversely, excessive biomechanical stress at spinal enthesis or peripheral enthesis with mechanically related or degenerative conditions is associated with a normal immune system attempts at cytokine fine tuning, but in this setting, it is commensurate to sustained abnormal biomechanical stressing. Unlike SpA, where restoration of aberrant and excessive cytokine “fine tuning” is efficacious, antagonism of these pathways in biomechanically related disease may be of limited or even no value.

Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Regulating the activity of matrix metalloproteinases (MMPs) is a potential strategy for osteoarthritis (OA) therapy, although delivering this effect in a spatially and temporally localised fashion remains a challenge. Here, we report an injectable and self-healing hydrogel enabling factor-free MMP regulation and biomechanical competence in situ. The hydrogel is realised within one minute upon room temperature coordination between hyaluronic acid (HA) and a cell-friendly iron-glutathione complex in aqueous environment. The resultant gel displayed up to 300% in shear strain and tolerance towards ATDC 5 chondrocytes, in line with the elasticity and biocompatibility requirements for connective tissue application. Significantly enhanced inhibition of MMP-13 activity was achieved after 12 hours in vitro, compared with a commercial HA injection (OSTENIL PLUS). Noteworthy, 24-hour incubation of a clinical synovial fluid sample collected from a late-stage OA patient with the reported hydrogel was still shown to downregulate synovial fluid MMP activity (100.0 +/- 17.6 % --> 81.0 +/- 7.5 %), with at least comparable extent to the case of the OSTENIL PLUS-treated SF group (100.0 +/- 17.6 % --> 92.3 +/- 27.3 %). These results therefore open up new possibilities in the use of HA as both mechanically-competent hydrogel as well as a mediator of MMP regulation for OA therapy.

Osteoarthritis (OA) is a heterogeneous disease in which the cross-talk between the cells from different tissues within the joint is affected as the disease progresses. Extracellular vesicles (EVs) are known to have a crucial role in cell-cell communication by means of cargo transfer. Subchondral bone (SB) resident cells and its microenvironment are increasingly recognised to have a major role in OA pathogenesis. The aim of this study was to investigate the EV production from OA SB mesenchymal stromal cells (MSCs) and their possible influence on OA chondrocytes. Small EVs were isolated from OA-MSCs, characterized and cocultured with chondrocytes for viability and gene expression analysis, and compared to small EVs from MSCs of healthy donors (H-EVs). OA-EVs enhanced viability of chondrocytes and the expression of chondrogenesis-related genes, although the effect was marginally lower compared to that of the H-EVs. miRNA profiling followed by unsupervised hierarchical clustering analysis revealed distinct microRNA sets in OA-EVs as compared to their parental MSCs or H-EVs. Pathway analysis of OA-EV miRNAs showed the enrichment of miRNAs implicated in chondrogenesis, stem cells, or other pathways related to cartilage and OA. In conclusion, OA SB MSCs were capable of producing EVs that could support chondrocyte viability and chondrogenic gene expression and contained microRNAs implicated in chondrogenesis support. These EVs could therefore mediate the cross-talk between the SB and cartilage in OA potentially modulating chondrocyte viability and endogenous cartilage regeneration.

Entheseal spinal inflammation and new bone formation with progressive ankylosis may occur in ankylosing spondylitis (AS) and psoriatic arthritis (PsA). This study evaluated whether JAK inhibition with tofacitinib modulated the key disease associated cytokines, TNF and IL-17A and whether tofacitinib also modulated bone marrow stromal cell-derived mesenchymal stem cells (MSCs) function including osteogenesis, since post inflammation new bone formation occurs in these conditions. Methods: Conventional entheseal derived αβ CD4+ and CD8+ T-cells were in-vestigated following anti-CD3/CD28 bead stimulation to determine IL-17A and TNF levels in Tofacitinib treated (1000nM) peri-entheseal bone (PEB) and peripheral blood mononuclear cells (PBMC) following ELISA. Bone marrow stromal cell-derived mesenchymal stem cells (MSCs) colony forming unit (CFU-F) and multilineage potential was evaluated using tofacitinib (dosag-es ranging between 100, 500, 1000 and 10000nM). Results: Induced IL-17A and TNF cytokine production from both entheseal CD4+ T-cells and CD8+ T-cells were effectively inhibited by to-facitinib. Tofacitinib treatment did not impact on CFU-F potential or in vitro chondro- and oste-ogenesis. However, tofacitinib stimulation increased MSC adipogenic potential with greater Oil Red O stained area. Conclusion: Inducible IL-17A and TNF production by healthy human enthe-seal CD4+ and CD8+ T-cells was robustly inhibited in vitro by tofacitinib. However, tofacitinib did not impact on MSC osteogenesis but stimulated in vitro MSC adipogenesis, the rele-vance of which needs further evaluation given the adipocytes are associated with new bone formation in SpA.

Osteoarthritis (OA) is a disease of the whole joint organ, characterized by the loss of cartilage, and structural changes in bone including the formation of osteophytes, causing disability and loss of function. It is also associated with systemic mediators and low-grade inflammation. Currently, there is negligible/no availability of specific biomarkers that can be used to facilitate the diagnosis and treatment of OA. The most unmet clinical need is, however, related to the monitoring of disease progression over a short period that can be used in clinical trials. In this review, the value of biomarkers identified over the past decade has been highlighted. These biomarkers are associated with the synthesis and breakdown of cartilage, including collagenous and noncollagenous biomarkers, inflammatory and anti-inflammatory biomarkers, expressed in the biological fluid such as serum, synovial fluid, and urine. Broad validation of novel and clinically applicable biomarkers and their involvement in the pathways are particularly needed for early-stage diagnosis, monitoring disease progression, and severity and examining new drugs to mitigate the effects of this highly prevalent and debilitating condition.

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.

[Abstract] Osteoarthritis (OA) is the most common musculoskeletal disorder. Although joint replacement remains the standard of care for knee OA patients, knee joint distraction (KJD), which works by temporarily off-loading the joint for 6–8 weeks, is becoming a novel joint-sparing alternative for younger OA sufferers. The biological mechanisms behind KJD structural improvements remain poorly understood but likely involve jointresident regenerative cells including multipotent stromal cells (MSCs). In this study, we hypothesized that KJD leads to beneficial cartilage-anabolic and anti-catabolic changes in joint-resident MSCs and investigated gene expression profiles of synovial fluid (SF) MSCs following KJD as compared with baseline. To obtain further insights into the effects of local biomechanics on MSCs present in late OA joints, SF MSC gene expression was studied in a separate OA arthroplasty cohort and compared with subchondral bone (SB) MSCs from medial (more loaded) and lateral (less loaded) femoral condyles from the same joints. In OA arthroplasty cohort (n = 12 patients), SF MSCs expressed lower levels of ossification- and hypotrophy-related genes [bone sialoprotein (IBSP), parathyroid hormone 1 receptor (PTH1R), and runt-related transcription factor 2 (RUNX2)] than did SB MSCs. Interestingly, SF MSCs expressed 5- to 50-fold higher levels of transcripts for classical extracellular matrix turnover molecules matrix metalloproteinase 1 (MMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and tissue inhibitor of metalloproteinase-3 (TIMP3), all (p < 0.05) potentially indicating greater cartilage remodeling ability of OA SF MSCs, compared with SB MSCs. In KJD cohort (n = 9 patients), joint off-loading resulted in sustained, significant increase in SF MSC colonies’ sizes and densities and a notable transcript upregulation of key cartilage core protein aggrecan (ACAN) (weeks 3 and 6), as well as reduction in pro-inflammatory C–C motif chemokine ligand 2 (CCL2) expression (weeks 3 and 6). Additionally, early KJD changes (week 3) were marked by significant increases in MSC chondrogenic commitment markers gremlin 1 (GREM1) and growth differentiation factor 5 (GDF5). In combination, our results reveal distinct transcriptomes on joint-resident MSCs from different biomechanical environments and show that 6- week joint off-loading leads to transcriptional changes in SF MSCs that may be beneficial for cartilage regeneration. Biomechanical factors should be certainly considered in the development of novel MSC-based therapies for OA. FOREUM: Foundation for Research in Rheumatology; 104929

The concentration of bone marrow (BM) aspirate (BMA) is increasingly valued for bone and cartilage repair, particularly with the rarity and donor-variability of BM-multipotential stromal cells (BM-MSCs). The present study aimed to assess BM-MSC yield following BM concentration using a fast and compact-sized vertical centrifugation system. BMA concentrate (BMAC) was separated in a 1 min process and collected easily after an automatic discarding of plasma and red blood cells. A significant increase in CD45low CD271high cells per BMAC volume (measured using flow-cytometry) was noted (4-fold, p = 0.0001). Additionally, the vertical centrifugation system helped to enrich colony numbers (assessed by CFU-F assays) in BMAC comparably with conventional centrifugation systems, BioCUE™ and SmartPReP-2® (4.3-fold, 4.6-fold and 3-fold, respectively). Next, a functional assessment of BM-MSCs processed by vertical centrifugation was performed, and MSC viability and proliferation were not affected. Also, these BM-MSCs showed similar alkaline phosphatase and calcium levels to those of BMA-MSCs when osteogenically induced. Furthermore, glycosaminoglycans and Nile red levels in addition to the gene expression assays confirmed that there was no significant change in chondrogenic or adipogenic abilities between BMA-MSCs and BMAC-MSCs. The expression levels of selected angiogenic and immunomodulatory mediators were also similar between the two groups. Collectively, the vertical centrifugation system helped to enrich BM-MSCs effectively, while maintaining cell viability and functions. Thus, such a vertical centrifugation system for BM concentration can be valuable for various regenerative therapies.

The current management of critical size bone defects (CSBDs) remains challenging and requires multiple surgeries. To reduce the number of surgeries, wrapping a biodegradable fibrous membrane around the defect to contain the graft and carry biological stimulants for repair is highly desirable. Poly(&epsilon, caprolactone) (PCL) can be utilised to realise nonwoven fibrous barrier-like structures through free surface electrospinning (FSE). Human periosteum and induced membrane (IM) samples informed the development of an FSE membrane to support platelet lysate (PL) absorption, multipotential stromal cells (MSC) growth, and the prevention of cell migration. Although thinner than IM, periosteum presented a more mature vascular system with a significantly larger blood vessel diameter. The electrospun membrane (PCL3%-E) exhibited randomly configured nanoscale fibres that were successfully customised to introduce pores of increased diameter, without compromising tensile properties. Additional to the PL absorption and release capabilities needed for MSC attraction and growth, PCL3%-E also provided a favourable surface for the proliferation and alignment of periosteum- and bone marrow derived-MSCs, whilst possessing a barrier function to cell migration. These results demonstrate the development of a promising biodegradable barrier membrane enabling PL release and MSC colonisation, two key functionalities needed for the in situ formation of a transitional periosteum-like structure, enabling movement towards single-surgery CSBD reconstruction.

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 (&alpha, and &beta, ) stimulation. Fluorescence-activated cell sorting was used to purify uncultured BM-MSCs from younger (19&ndash, 41, n = 6) and older (59&ndash, 89, n = 6) donors based on the CD45lowCD271+ phenotype, and hematopoietic-lineage cells (BM-HLCs, CD45+CD271&minus, ) 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 IFN&beta, 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.

[Abstract] Background: The biological mechanisms that contribute to atrophic long bone non-union are poorly understood. Multipotential mesenchymal stromal cells (MSCs) are key contributors to bone formation and are recognised as important mediators of blood vessel formation. This study examines the role of MSCs in tissue formation at the site of atrophic non-union. Materials and Methods: Tissue and MSCs from non-union sites (n = 20) and induced periosteal (IP) membrane formed following the Masquelet bone reconstruction technique (n = 15) or bone marrow (n = 8) were compared. MSC content, differentiation, and influence on angiogenesis were measured in vitro. Cell content and vasculature measurements were performed by flow cytometry and histology, and gene expression was measured by quantitative polymerase chain reaction (qPCR). Results: MSCs from non-union sites had comparable differentiation potential to bone marrow MSCs. Compared with induced periosteum, non-union tissue contained similar proportion of colony-forming cells, but a greater proportion of pericytes (p = 0.036), and endothelial cells (p = 0.016) and blood vessels were more numerous (p = 0.001) with smaller luminal diameter (p = 0.046). MSCs showed marked differences in angiogenic transcripts depending on the source, and those from induced periosteum, but not non-union tissue, inhibited early stages of in vitro angiogenesis. Conclusions: In vitro, non-union site derived MSCs have no impairment of differentiation capacity, but they differ from IP-derived MSCs in mediating angiogenesis. Local MSCs may thus be strongly implicated in the formation of the immature vascular network at the non-union site. Attention should be given to their angiogenic support profile when selecting MSCs for regenerative therapy.

Treatment of fractures remains challenging and carries a high economical burden to both patients and society. In order to prevent some of the complications, the use of bone adhesives has been proposed, but up to date, bone adhesives are not part of the current clinical practice. Early results of use of bone cements and bone glues are promising, focusing in the areas of highly fragmented fractures, fixation of long bone fractures, filling bone voids and defects, promoting osseointegration, preventing non-union while maintaining the reduction of fracture fixation. This review aims to describe the state-of-the-art of the development, properties and use of adhesives in fracture treatment.

The biology of mesenchymal stem cells (MSCs) in humans is incompletely understood and a possible role of systemically circulating cells in health and autoimmune disease remains controversial. Physiological movement of bone marrow MSCs to sites of injury would support the rationale for intravenous administration for relocation to damaged organs. We hypothesized that biophysical skeletal trauma rather than molecular cues may explain reported MSC circulation phenomena. Deep-femoral vein (FV) and matched peripheral vein blood samples (PVBs) were collected from patients undergoing lower-limb orthopaedic procedures during surgery (tibia using conventional sequential reaming, n = 9, femur using reamer/irrigator/aspirator (RIA), n = 15). PVBs were also taken from early (n = 15) and established (n = 12) rheumatoid arthritis (RA) patients and healthy donors (n = 12). Colony-forming unit-fibroblasts (CFU-Fs) were found in 17/36 FVBs but only 7/74 PVBs (mostly from femoral RIA), highly proliferative clonogenic cells were not generated. Only one colony was found in control/RA samples (n = 28). The rare CFU-Fs&rsquo, MSC nature was confirmed by phenotypic: CD105+/CD73+/CD90+ and CD19-/CD31-/CD33-/CD34-/CD45-/CD61-, and molecular profiles with 39/80 genes (including osteo-, chondro-, adipo-genic and immaturity markers) similar across multiple MSC tissue controls, but not dermal fibroblasts. Analysis of FVB-MSCs suggested that their likely origin was bone marrow as only two differences were observed between FVB-MSCs and IC-BM-MSCs (ACVR2A, p = 0.032 and MSX1, p = 0.003). Stromal cells with the phenotype and molecular profile of MSCs were scarcely found in the circulation, supporting the hypothesis that their very rare presence is likely linked to biophysical micro-damage caused by skeletal trauma (here orthopaedic manipulation) rather than specific molecular cues to a circulatory pool of MSCs capable of repair of remote organs or tissues. These findings support the use of organ resident cells or MSCs placed in situ to repair tissues rather than systemic administration.

Background: Mesenchymal stem cells (MSCs) have emerged as a promising candidate for tissue regeneration and restoration of intra-articular structures such as cartilage, ligaments, and menisci. However, the routine use of MSCs is limited in part by their low numbers and the need for methods and procedures outside of the joint or surgical field. Purpose: To demonstrate feasibility of a technique in which minimally manipulated synovial MSCs can be mobilized during knee arthroscopy, thereby showing proof of concept for the future evaluation and clinical use of native joint resident MSCs in single-stage joint repair strategies. Study Design: Descriptive laboratory study. Methods: Patients (n = 15) undergoing knee arthroscopy who were free from synovitis or active inflammation were selected. Three samples of irrigation fluid were collected from each patient at inception of the procedure, after an initial inspection of the joint, and after agitation of the synovium. MSC numbers were evaluated by colony forming unit–fibroblastic assay. The phenotype of synovial fluid resident and synovial-mobilized MSCs was determined by flow cytometry, and their functionality was determined by trilineage differentiation. Adhesion of culture-expanded mobilized MSCs to fibrin scaffolds was also evaluated to ascertain whether mobilized MSCs might concentrate at sites of bleeding. Results: Normal irrigation during arthroscopy depleted resident synovial fluid MSCs (4-fold decrease, n = 15). Numbers of MSCs mobilized through use of a purpose-made device were significantly higher (105-fold) than those mobilized through use of a cytology brush (median of 5763 and 54 colonies, respectively; P = .001; n = 15). The mobilized cellular fraction contained viable MSCs with proliferative potential and trilineage differentiation capacity for bone, cartilage, and fat lineages, and cultured daughter cells exhibited the standard MSC phenotype. Following culture, mobilized synovial MSCs also adhered to various fibrin scaffolds in vitro. The technique was simple and convenient to use and was not associated with any complications. Conclusion: Numbers of functional MSCs can be greatly increased during arthroscopy through use of this technique to mobilize cells from the synovium. Clinical Relevance: This study highlights a novel, single-stage technique to increase joint-specific, synovial-derived MSCs and thereby increase the repair potential of the joint. This technique can be undertaken during many arthroscopic procedures, and it supports the principle of integrating mobilized MSCs into microfracture sites and sites of bleeding or targeted repair through use of fibrin-based and other scaffolds.

Background: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. Hypothesis: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. Study Design: Controlled laboratory study. Methods: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques—BM aspiration and BM collection—after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. Results: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45–CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56– cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. Conclusion: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. Clinical Relevance: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.

The role of native (not culture-expanded) joint-resident mesenchymal stem cells (MSCs) in the repair of joint damage in osteoarthritis (OA) is poorly understood. MSCs differ from bone marrow-residing haematopoietic stem cells in that they are present in multiple niches in the joint, including subchondral bone, cartilage, synovial fluid, synovium and adipose tissue. Research in experimental models suggests that the migration of MSCs adjacent to the joint cavity is crucial for chonodrogenesis during embryogenesis, and also shows that synovium-derived MSCs might be the primary drivers of cartilage repair in adulthood. In this Review, the available data is synthesized to produce a proposed model in which joint-resident MSCs with access to superficial cartilage are key cells in adult cartilage repair and represent important targets for manipulation in 'chondrogenic' OA, especially in the context of biomechanical correction of joints in early disease. Growing evidence links the expression of CD271, a nerve growth factor (NGF) receptor by native bone marrow-resident MSCs to a wider role for neurotrophins in OA pathobiology, the implications of which require exploration since anti-NGF therapy might worsen OA. Recognizing that joint-resident MSCs are comparatively abundant in vivo and occupy multiple niches will enable the optimization of single-stage therapeutic interventions for OA.

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.

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 CD45lowCD271high 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 CD45lowCD271high cell counting in bone marrow aspirates, bone marrow concentrates, and samples loaded onto a collagen scaffold. The CD45lowCD271high 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 CD45lowCD271high 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 CD45lowCD271high 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 CD45lowCD271high 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 CD45lowCD271high 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 CD45lowCD271high cells may provide a useful measurement of bone marrow quality. While the specificity of this measurement of CD45lowCD271high cells remained low in our experimental conditions, CD45lowCD271high 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 CD45lowCD271high cell counting may be a useful tool for improving the quality of regenerative therapy.

Background:Synovial fluid contains resident mesenchymal stem cells (SF-MSCs) that are derived from the synovial membrane and may interact with superficial cartilage injury sites. We previously reported on a novel methodology for increasing the number of MSCs in the knee joints using synovial brushing combined with platelet lysate (PL) as a chondrogenic inducer [1, 2].Objectives:The purpose of this study was to evaluate autologous and allogenic PL as a chondrogenic inducer and the chondrogenic potential of the mobilised MSCs without further ex vivo expansion. The desired goal of the study was to provide in vitro proof of concept of direct chondrogensis without resort to MSC expansion protocols, since adequate MSCs towards repair could be mechanically procured in a minimally invasive fashion.Methods:SF-MSCs were derived from the joint cavity of patients undergoing arthroscopy procedures. For the mechanical release of MSCs ‘before’ and ‘after’ brushing the synovium with the novel device (Figure 1A), samples of irrigation fluid were collected and MSC numbers were evaluated by CFU-F assay and flow cytometry for stromal and immune populations. Standard chondrogenic assay was performed on uncultured and cultured expanded synovial MSCs. Pellet cultures were maintained in complete chondrogenic media (CCM), DMEM+50% autologous filtared platelet concentrate (fPC), 50% Stemulate (allogeneic human PL; Cook Regentec, Indianapolis, IN), or expansion media (control). Chondrogenesis was assessed by Glycosaminoglycan (GAG) andToluidine bluestaining. Autologous blood was processed through a gravity-based filtration system, HemaTrate®(HT; Cook Regentec, Indianapolis, IN), to produce a PC.Figure 1.Flow cytometry analysis of stromal and immune populations before’ and ‘after’ mechanically release of synovial with the novel device (CD90highCD45Lowin red circle) (A). Uncultured synovial cells after 21 days exposure to complete chondrogenic media Toulid Blue staining (B) Gags level (C) n=3.Results:Mechanically mobilized SF-MSC numbers increased as measured by CFU-F assay and flow cytometry for CD90HighCD45Lowcells (p+HLA-DR+CD206+CD86+M2 macrophages also increased (p0.5). Uncultured synovial cells produced significantly more GAG when cultured in CCM or DMEM + 50% autologous PC compared to control (pConclusion:Synovial MSCs can be mechanically released in sufficient number to undergo in vitro chondrogenic induction with significant chondrogenic activity without the need for ex vivo culture expansion. In vitro, autologous PC can be used as chondrogenic inducer for uncultured SF-MSCs. The data presented here supports one stage arthroscopy procedures for cartilage repairReferences:[1]T.G. Baboolal, S.C. Mastbergen, E. Jones, S.J. Calder, F.P. Lafeber, D. McGonagle, Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction, Annals of the rheumatic diseases 75(5) (2016) 908-15.[2]A. Altaie, T.G. Baboolal, O. Wall, E. Jones, D. McGonagle, Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use, Cytotherapy 20(3) (2018) 375-384.Disclosure of Interests:Ala Altaie: None declared, Elena Jones: None declared, Owen Wall: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

Background & Aim Synovial fluid contains resident mesenchymal stem cells (SF-MSCs) that are derived from the synovial membrane and may interact with superficial cartilage injury sites . We previously reported novel methodology for increasing the number of MSC in knee joints using synovial brushing and of platelet lysate (PL) as chondrogenic inducer [1, 2]. The purpose of this study was to evaluate autologous and allogenic PL as chondrogenic inducer and the chondrogenic potential of the mobilised MSCs without resort to in vitro expansion. Methods, Results & Conclusion SF-MSCs were derived from joint cavity of patients undergoing arthroscopy procedures.Autologous blood was processed through platelet concentrating device HemaTrate (HT) to produce platelets concentrate (PC). For the mechanical release of MSCs‘before’ and ‘after’ brusing samples of irrigation fluid were collected and MSCs numbers were evaluated CFU-F assay and flow cytomery.Standard chondrogenic assay was performed on uncultured and cultured synovial cells. Pellet cultures were maintained in complete chondrogenic media (CCM), DMEM+50% of autologous PC,50% Stemulate (allogeneic PL) or expansion media (control). Chondrogenesis was assessed by Glycosaminoglycan (GAG) and Toluidine blue staining. The HT device significantly concentrated platelets and WBCs by 6 P 0.5. Unculturedsynovial cells produced significantly more GAG when cultured in CCM or DMEM+50% autologous PC compared to control P Conclusion: Autologous PC can be used as chondrogenic inducer for uncultured SF-MSCs. Sufficient cells can be released mechanically to be subjected to chondrogenic induction with significant chondrogenic activity without the need of culture expansion. This suggests possibility of one stage arthroscopy procedures for cartilage repair.

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-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low 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.

Mesenchymal stromal cells (MSCs) possess self-renewal and multilineage differentiation potential, indicating their prospects as cellular therapeutic agents for regenerative medicine. Although adult bone marrow (BM) is the major source of these cells for clinical use, harvesting requires invasive procedures. Therefore, alternative sources, such as peripheral blood (PB), are needed. The objective of the current study was to compare PB-MSCs and BM-MSCs with regard to their biological characteristics. PB-MSCs and BM-MSCs were isolated from 4-week-old BALB/c white mice by density gradient centrifugation and cultured in DMEM + 10% fetal bovine serum until passage four. Morphological features, proliferation, cell surface marker expression and trilineage differentiation potential were assessed for both PB-MSCs and BM-MSCs. No significant differences in morphological features were observed. BM-MSCs had a higher proliferative capability than PB-MSCs as measured by XTT assays. Both PB-MSCs and BM-MSCs had broadly similar cell surface marker expression, but PB-MSCs had positive expression of cluster of differentiation (CD)146 and CD140b. Both PB-MSCs and BM-MSCs were capable of trilineage differentiation. Although BM-MSCs had a greater capacity for osteogenic and chondrogenic differentiation than PB-MSCs, PB-MSCs had a better capability for adipogenic differentiation than BM-MSCs. In conclusion, PB-MSCs and BM-MSCs have very similar biological characteristics. Thus, PB is a promising source for easily obtaining MSCs in mice.

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 CD45lowCD271+ 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 (r=−0.527, p<0.0001) or flow cytometry (r=−0.307, p=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, p<0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, p<0.0001, and 27%, p<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 CD45lowCD271+ MSCs from young and old donors (n=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 CD45lowCD271+ 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.

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.

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

Background Osteoarthritis (OA) is the most common degenerative joint disorder, mainly afflicting the weight–bearing joints, and is the leading cause of physical disability worldwide. Despite the identified risk factors, the exact pathogenesis of osteoarthritis remains unclear (1). Formation of mesenchymal stem cells’ (MSC) clusters and their aberrant osteogenic differentiation in OA subchondral bone (SB) has been proposed as a key contributor to progression of OA in animal models (2) and in human hip OA (3). MSCs have a crucial role in joint repair but it remains unknown how OA severity affects MSC numbers or characteristics. Knee OA provides a good model to determine this as in knee OA, distribution of damage is usually asymmetrical and tends to be more severe in the medial (Med) compared to the lateral (Lat) compartment (4). Objectives The aim of this study was to determine whether there were differences in MSC numbers, topography and gene expression (GE) between Med and Lat femoral condyles of patients with knee OA. Methods Condyles were obtained from OA patients that underwent total knee replacement (n=16; UK Ethics Committee approval, 14/YH/0087). Decalcified samples were histologically evaluated for cartilage damage, bone sclerosis and CD271+ MSC distribution (2). MSCs were extracted from SB and sorted using the CD271+CD45- phenotype for GE analysis. Colony forming unit (CFU-F) (3), trilineage differentiation (5) and population doubling (PD) assays were performed. MSC numbers, topography and GE were compared between condyles. Results Med condyles presented significantly higher (p Conclusion Upregulation of ossification-related genes in Med condyle MSCs suggest their potential contribution to sclerotic plate formation and OA severity. Further work is needed to establish if biomechanical or biological stimulation of these MSCs can result in GE modulation in preference of cartilage tissue formation and subchondral bone restoration. References [1] Conaghan. Nat Rev Rheumatol (2013). [2] Zhen. Nat Med (2013); [3] Campbell. Arthritis Rheumatol (2016); [4] Bae. Cartilage (2010). [5] Altaie. Cytotherapy (2018); [6] Hu. Mol Med Rep (2017); [7] Garcia-Martin. Mol Endocrinol (2014); [8] Chiellini. Biochem Biophys Res Commun (2008) Acknowledgement CSR funded by fellowship Xunta de Galicia (Consell de Cul, Educ e Ordenacion Univ). TB and DM part-supported by NIHR Leeds Musculoskeletal Biomed Research Centre. TB part-supported by FOREUM. JE part-funded by AO foundation start-up grant. Disclosure of Interests Clara Sanjurjo Rodriguez: None declared, Thomas Baboolal: None declared, Agata Burska: None declared, Frederique Ponchel: None declared, Jehan El-Jawhari: None declared, Joseph Aderinto: None declared, Owen Wall: None declared, Hemant Pandit Grant/research support from: GSK, Consultant for: For education for Bristol Myers Squibb, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB, Elena Jones: None declared

Barrier membranes are popularly used for guided bone regeneration (GBR). However, more knowledge is needed to assess how these membranes could be of therapeutic value when populated with native multipotent stromal cells (MSCs), particularly in the orthopaedic field. The present manuscript investigated the activities of human bone marrow-multipotent stromal cells (BM-MSCs) when loaded on to two differently structured pure collagen membranes. A crosslinked collagen membrane (CS) was tested versus a non-crosslinked bilayer collagen membrane, Bio-Gide® (BG). Following loading with BM aspirate containing native MSCs, cell attachment to the membranes was examined using electron microscopy and flow cytometry. Furthermore, alkaline phosphatase (ALP) expression and calcium deposition levels were investigated for these BM-aspirate-loaded membranes. Culture-expanded BM-MSCs were also used to load membranes and confirm the MSC functional data. All membranes supported BM-MSC attachment. However, larger numbers of attached BM-MSCs were detected for CS as compared to BG (p = 0.0010). In osteogenic medium, ALP activity was higher for CS than BG (p = 0.0312). Total calcium deposition (not normalised to cell count) was also higher for CS than BG (p = 0.0073). Consistently, the normalised secreted vascular endothelial growth factor A (VEGF-A) levels were higher in BM-MSCs loaded on CS relative to BG (p = 0.0302). Collectively, both collagen membranes supported the osteogenic functions of BM-MSCs. However, CS was found to be overall superior probably since it provided more BM-MSC attachment. These collagen membranes could potentially be used to improve GBR outcomes in orthopaedic applications.

Mesenchymal stem cells (MSC) from bone marrow and periosteum are known to be heavily involved in fracture repair and bone regeneration is thought to be impaired when the surrounding skeletal muscle is damaged. Recent literature from mouse in vivo models suggest that cells originating from skeletal muscle can occupy a fracture callus during open fracture repair when periosteum is compromised. This systematic review set out to ascertain whether there are MSCs residing in human skeletal muscle and whether cells from human skeletal muscle are capable of forming bone in vitro and in vivo. Original journal articles were selected if they included the terms "skeletal muscle" and "mesenchymal" and used human skeletal muscle samples. Between January 2005 and September 2016, 1000 articles were screened of which, 16 studies met the inclusion criteria for this review. Human skeletal muscle derived cells (SMDC) had the MSC phenotype, positive for CD73, CD90 and CD105 and negative for CD34 and CD45 as well as the potential to differentiate into osteoblasts, chondrocytes and adipocytes in vitro. In addition, SMDC could form bone in vivo when seeded onto an osteoinductive scaffold. A subset of SMDC expressing a pericyte marker (PDGFRα) also expressed the MSC phenotype and were more osteogenic in vivo in comparison to SMDC expressing a satellite cell marker (CD56). The studies included were limited through variation of SMDC extraction methods and tissue culture conditions, which causes heterogeneuous cell cultures. Also, in vitro differentiation assays were not always carried out with bone marrow MSC positive controls. Current evidence suggests that cells with the MSC phenotype reside within human skeletal muscle and are capable of in vivo bone formation in combination with osteoinductive bone scaffolds. This has implications of future development of guided bone regeneration strategies to enhance large bone defect repair, whereby more thought into whether the fracture site should be "blocked" from the skeletal muscle should be carried out.

Objective: In patients with osteoarthritis (OA), bone marrow lesions (BMLs) are intimately linked to disease progression. We hypothesized that aberrant multipotential stromal cell (also known as mesenchymal stem cell [MSC]) responses within bone tissue contributes to BML pathophysiology. The aim of this study was to investigate BML and non-BML native subchondral bone MSCs for numeric, topographic, in vitro functional, and gene expression differences. Methods: Ex vivo 3T magnetic resonance imaging (MRI) of the femoral heads of 20 patients with hip OA was performed. MRI-determined BML and non-BML regions were excised and enzymatically treated to extract cells and quantify MSCs using flow cytometry and colony-forming unit–fibroblast (CFU-F) assay. Immunohistochemical analysis was performed to determine in vivo CD271+ MSC distribution. Culture-expanded CD271+ cells were analyzed for tripotentiality and gene expression. Results: BML regions were associated with greater trabecular bone area and cartilage damage compared with non-BML regions. The proportion of CD45−CD271+ MSCs was higher in BML regions compared with non-BML regions (median difference 5.6-fold; P < 0.001); the CFU-F assay showed a similar trend (median difference 4.3-fold; P = 0.013). Immunohistochemistry revealed CD271+ cell accumulation in bone adjacent to cartilage defects and areas of osteochondral angiogenesis. BML MSCs had lower proliferation and mineralization capacities in vitro and altered expression of TNFSF11/RANKL and CXCR4/stromal cell–derived factor 1 receptor. OA MSCs showed up-regulated transcripts for CXCR1 and CCR6 compared with MSCs derived from healthy or osteoporotic bone. Conclusion: This study is the first to show numeric and topographic alterations in native MSCs in the diseased bone of patients with hip OA. Given the associated functional perturbation of MSCs, these data suggest that subchondral bone MSC manipulation may be an OA treatment target.

Background:Fat formation in the bone adjacent to the enthesis is an important but poorly characterised intermediate stage in new bone formation that occurs in the spine in AS. We and others have previously reported that IL-17A can increase mesenchymal stem cell (MSC) mediated osteogenesis in normal and AS spinal tissue (1, 2).Objectives:Herein we investigate the impact of IL-17A & TNF on MSC adipogenesis from spinal enthesis tissue.Methods:Samples from healthy spinous process and interspinous ligament (n=14, median age = 53) were separated into the peri-entheseal bone (PEB) and entheseal soft tissue (EST) & enzymatically digested. Minimally passaged (Results:EST MSCs have a significantly higher adipogenic potential than matched PEB MSCs (n=14, pConclusion:Given the inverse link between MSC mediated osteogeneis & adipogenesis, these findings reveal a role of IL-17A especially on EST MSCs. The rapid formation of adipocytes seen in EST MSCs may be relevant to MRI determined peri-entheseal bone “shiny corners” due to post inflammation fat accumulation. Elevated transcripts associated with pre-adipocytes & undifferentiated MSCs support the idea of plasticity between early osteogenesis & adipogenesis. Downregulation of transcripts for proteins associated with protection against lipolysis allows for the rationalising of the gradual loss of the shiny corners seen in AS preceding subsequent new bone formation.References:[1]RUSSELL, T., A. WATAD, C. BRIDGEWOOD, A. KHAN, A.S. RAO, P. LOUGHENBURY, P. MILNER, R. DUNSMUIR, T. BABOOLAL, E. JONES, R. CUTHBERT and D. MCGONAGLE. IL-17A Induces Distinct Functional Differences Between Two Novel Mesenchymal Stem Cell Populations Identified at the Human Enthesis.Arthritis Rheumatol, 2019, 71 Suppl 10, pp.1-5362.[2]JO, S., S.E. WANG, Y.L. LEE, S. KANG, B. LEE, J. HAN, I.H. SUNG, Y.S. PARK, S.C. BAE and T.H. KIM. IL-17A induces osteoblast differentiation by activating JAK2/STAT3 in ankylosing spondylitis.Arthritis Res Ther, 2018, 20(1), p.115.[3]AHMED, M. and S.L. GAFFEN. IL-17 in obesity and adipogenesis.Cytokine Growth Factor Rev, 2010, 21(6), pp.449-53.[4]HANSEN, J.S., S. DE MARE, H.A. JONES, O. GORANSSON and K. LINDKVIST-PETERSSON. Visualization of lipid directed dynamics of perilipin 1 in human primary adipocytes.Sci Rep, 2017, 7(1), p.15011.Disclosure of Interests:Tobias Russell Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Charlie Bridgewood: None declared, Almas Khan: None declared, Abhay S Rao: None declared, Peter Loughenbury: None declared, Peter Millner: None declared, Robert Dunsmuir: None declared, Ala Altaie: None declared, Elena Jones: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

Objective OA subchondral bone is a key target for therapy development. Osteocytes, the most abundant bone cell, critically regulate bone formation and resorption. Their progenitors, mesenchymal stem cells (MSCs), display altered behaviour in osteoarthritic subchondral bone. This study investigated the relationships between native osteocytes and native MSCs in osteoarthritic femoral heads. Methods To avoid culture manipulations, a bone treatment procedure was developed to simultaneously obtain pure osteocyte-enriched fragments and matched native CD45-CD271+ MSCs. Gene expression in osteocytes and MSCs was compared between healthy and OA bone and selected molecules were examined by immunohistochemistry in relation to OA tissue pathology. Cell sorting and standard trilineage differentiation assays were employed to test OA MSC functionality. Results Native osteocyte enrichment was confirmed histologically and by higher-level osteocyte maturation transcripts expression, compared with purified MSCs. Compared with healthy bone, native OA osteocytes expressed 9- and 4-fold more early/embedding osteocyte molecules E11 and MMP14, and 6-fold more osteoprotegerin (P Conclusions In human late-stage OA, osteogenically-committed MSCs and adjacent immature osteocytes exhibit a marked accumulation in sclerotic areas. This hitherto unappreciated MSC-early osteocyte axis could be key to understanding bone abnormalities in OA and represents a potential target for novel therapy development in early disease.

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.

The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion.

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.

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. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016.

Objectives Knee joint distraction (KJD) is a novel, but poorly understood, treatment for osteoarthritis (OA) associated with remarkable 'spontaneous' cartilage repair in which resident synovial fluid (SF) multipotential mesenchymal stromal cells (MSCs) may play a role. We hypothesised that SF hyaluronic acid (HA) inhibited the initial interaction between MSCs and cartilage, a key first step to integration, and postulate that KJD environment favoured MSC/cartilage interactions. Methods Attachment of dual-labelled SF-MSCs were assessed in a novel in vitro human cartilage model using OA and rheumatoid arthritic (RA) SF. SF was digested with hyaluronidase (hyase) and its effect on adhesion was observed using confocal microscopy. MRI and microscopy were used to image autologous dual-labelled MSCs in an in vivo canine model of KJD. SF-HA was investigated using gel electrophoresis and densitometry. Results Osteoarthritic-synovial fluid (OA-SF) and purified high molecular weight (MW) HA inhibited SF-MSC adhesion to plastic, while hyase treatment of OA-SF but not RA-SF significantly increased MSC adhesion to cartilage (3.7-fold, p9 MDa HA and this correlated with increases in adhesion (r=0.880). In the canine KJD model, MSC adhesion to cartilage was evident and also dependent on HA MW. Conclusions These findings highlight an unappreciated role of SF-HA on MSC interactions and provide proof of concept that endogenous SF-MSCs are capable of adhering to cartilage in a favourable biochemical and biomechanical environment in OA distracted joints, offering novel one-stage strategies towards joint repair.

Delayed bone healing and non-union occur in approximately 10% of long bone fractures. Despite intense investigations and progress in understanding the processes governing bone healing, the specific pathophysiological characteristics of the local microenvironment leading to non-union remain obscure. The clinical findings and radiographic features remain the two important landmarks of diagnosing non-unions and even when the diagnosis is established there is debate on the ideal timing and mode of intervention. In an attempt to understand better the pathophysiological processes involved in the development of fracture non-union, a number of studies have endeavoured to investigate the biological profile of tissue obtained from the non-union site and analyse any differences or similarities of tissue obtained from different types of non-unions. In the herein study, we present the existing evidence of the biological and molecular profile of fracture non-union tissue.

Background aims Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid–derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. Methods SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit–fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. Results PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. Conclusions This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage.

Background Nerve growth factor (NGF) is a key regulator of pain and anti-NGF therapy reduces osteoarthritis (OA) associated pain. However, anti-NGF therapy is associated with rapidly progressive OA (RPOA) [1]. In hip OA there is a 5-fold increase in mesenchymal stem cells (MSCs) from MRI bone marrow (BM) lesions, areas associated with OA progression [2]. MSCs in such lesions are uniformly positive for the NGF receptor, p75, which is also linked to chemotaxis and proliferation in other stromal cell compartments [3]. Objectives Evaluate anti-NGF treatment in monoiodoacetate (MIA) induced OA and test whether NGF influences human BM-MSC function. Methods Human tibial plateau (TP) bone was isolated from patients undergoing total knee replacement. OA was induced in male Wistar rats (n=6) by intra-articular injection of 0.3 mg MIA. Each animal was treated with subcutaneous injection of control human IgG or anti-NGF (3 mg/kg) at Days 5, 10 and 15. Human and animal tissues sections were prepared for histological analysis using H&E staining and immunohistochemistry (IHC) using anti-p75 and anti-NGF antibodies. BM-MSCs were isolated from iliac crest aspirates and cultured under normal conditions. Expression of p75 was induced following overnight incubation with 400mM ethanol (EtOH) and confirmed by flow cytometry. Proliferation was assessed ± EtOH and 0–1 μg/ml NGF. Results Regions adjacent to cartilage loss in human TP showed abundant stromal proliferation, NGF and p75 immunoreactivity. In MIA model, by Day 18 there was substantial loss of cartilage, bone remodelling and stromal proliferation mimicking human disease. By Week 4 animals demonstrated unequal weight-bearing (p To investigate increased p75 positivity in knee OA, we restored p75 expression loss in expanded cell, to 97% of BM-MSCs (n=3) using EtOH. Increased MSC proliferation was seen at Days 6 and 9 (26%, p=0.03 and 30%, p=0.01 increase respectively, n=7) for 1 μg/ml NGF in the presence of EtOH compared to control (no NGF). In cultures without EtOH induction (absent p75) NGF had no effect. Conclusions TP bone from OA patients and rat MIA-treated subchondral bone contains p75 positive staining in regions of cartilage destruction and associated NGF positivity. Anti-NGF treatment exacerbated MIA-induced OA and reduced NGF and p75 expression. In vitro, NGF increased BM-MSC proliferation, suggesting NGF may be involved in the stromal proliferation seen at sites of OA damage. Thus, NGF may regulate MSC function. Complete blockade represents a novel mechanism for accelerated joint destruction in OA. References Hochberg MC et al. Arthritis Rheumatol 2016. Campbell TM et al Arthritis Rheumatol 2016. Jiang Y, et al. Cartilag Arthritis Res Ther 2015. Disclosure of Interest T. Baboolal: None declared, S. Al Hinai: None declared, E. Jones: None declared, J. Reckless Employee of: Rxcelerate Ltd, M. Foster Consultant for: Levicept Ltd, R. Doyle Employee of: Tetrad Discovery Ltd, K. af Forselles Consultant for: Levicept Ltd, S. Westbrook Shareholder of: Levicept Ltd, Employee of: Levicept Ltd, D. McGonagle: None declared

Background Both synovial and related synovial fluid-derived mesenchymal stem cells (SF-MSC) are highly proliferative and chondrogenic and have direct access to superficial cartilage injuries [1]. Human platelet lysate (PL) is being increasingly used for joint regenerative approaches [2,3] but the biological basis for in vivo effect, if any is lacking. Objectives The study evaluated the effect of PL on adhesion, proliferation, migration and trilineage differentiation of synovial cavity derived MSCs in comparison to “gold standard” foetal calf serum (FCS) based stem cell media. Methods MSCs were derived from joint cavity washout in patients undergoing anterior cruciate ligament reconstruction or meniscal repairs. Cells were plated in StemMACS (FCS media) or with DMEM Supplemented with 10% Stemulate (PL media, Cook Regentec). Standard MSC proliferation, trilineage differentiation assays, and transwell migration assays were performed for both conditions. Results PL enhanced MSC colonies sizes (CFU-F) by 2.5 folds compared to FCS (p=0.0023, n=5). PL shortened MSC doubling times (p=0.0411, n=6). However, PL had no impact on CFU-F numbers (p=0.5973, n=5). In transwell migration assays, MSC migration was significantly increased toward 10%PL compared to 10%FCS (p=0.0286, n=6). Under standard chondrogenic induction conditions, PL-expanded MSC showed 30% more sulphated GAG (sGAG) production compared to FCS-expanded cells (n=4). In osteogenic and adipogenic induction, Alizarin red staining Ca++ assay and Nile Red showed no significant difference compared to FCS expanded cells (p=0.3429, n=4). Interestingly, replacing TGFbβ with 20% PL was as good as standard chondrogenic media and with increasing PL percentage sGAG production increased (n=4). In same time using PL with high glucose media in 1:1 ratio was efficient to produced sGAG as compared to negative control (media only) (p=0.0286, n=4, figure 1). Moreover, substituting FCS with PL in the osteogenic media resulted in increased Ca++ deposition in both PL and FCS expanded cells by 90% and 100% compared standard osteogenic media. Conclusions This is the first study that assessed joint cavity MSC responses to PL. All aspects of joint cavity derived MSC biology towards cartilage repair were augmented in vitro with PL based media over “gold standard” MSC media. This work provides biological plausibility for the combination of PL with joint resident MSC towards endogenous joint repair strategies. References Jones, E.A., et al., Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single-cell level. Arthritis Rheum, 2008. 58(6): p. 1731–40. Morito, T., et al., Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford), 2008. 47(8): p. 1137–43. Ben Azouna, N., et al., Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum. Stem Cell Res Ther, 2012. 3(1): p. 6. Disclosure of Interest None declared

Background Osteoarthritis is a disease of the whole joint, but subchodral bone marrow lesions (BMLs) closely correlate with disease progression [1]. We have previously shown that in OA femoral heads, subchondral bone multipotential stromal cells (MSCs) were 5-fold more abundant in MRI determined BML areas where they also showed reduced mineralisation capacity and altered RANKL expression compared to non-BML areas [2]. This offered novel insight into MSC involvement in the bone remodeling process in OA. Objectives The current study investigated a multi-lineage gene expression profile of native CD271+ MSCs from OA femoral heads focusing on osteogenesis and bone remodeling related genes compared to CD271+ MSCs from healthy trabecular bone. Methods Femoral heads were obtained from 17 OA patients undergoing total hip arthroplasty. Control samples included healthy iliac crest bone from 12 patients undergoing autograft harvesting for bone reconstruction and healthy femoral neck bone from 2 patients, extracted following core decompression surgery for avascular necrosis of the femoral head. CD271+ MSCs were extracted by enzymatic digestion and purified by FACS, as previously described [3, 4]. MSC cultures were generated by standard methods [4]. Quantitative real-time PCR was performed using TaqMan assays for 12 transcripts and gene expression was normalised to HPRT1 . Results Sorted CD271+ cells from both groups displayed gene expression profiles indicative of their steady-state osteogenic, adipogenic, bone remodeling and stromal-support functions (Table 1). Three out of 11 transcripts showed significant increases in OA: alkaline phosphatase, melanoma cell adhesion molecule and osteoprotegerin (OPG) (p Conclusions This is the first study to show gene expression alterations in uncultured CD271+ MSCs from OA patients. A notable increase in OPG is suggestive of MSCs9 bias towards inhibition of bone resorption in late hip OA and further incriminates the RANKL/OPG pathway, specifically in MSCs, in OA pathophysiology. Further studies are needed to define the role of native MSCs in lesion associated OA disease progression and to evaluate these as a possible target for treatment. References Goldring et al, Nat Rev Rheumatol. 2016;12:632. Campbell et al, Arthritis Rheumatol. 2016;68:1648-. Jones et al., Arthritis Rheum. 2010 Jul;62:1944. Churchman et al., Arthritis Rheum. 2012 Aug;64:2632. Churchman et al., Stem Cells Int, 2017 (in press). Disclosure of Interest None declared

Background Osteoimmunology is an evolving field where the multipotential stromal cells (MSCs) can be considered as an important player linking immune response with bone generation. A group of pro-inflammatory cytokines including IFN-γ, TNF-α, IL-17 and IL-1, has been proven to have a licensing effect on MSCs promoting the immunomodulatory activities of MSCs (1). Importantly, these cytokines can regulate the osteogenic differentiation capability of MSCs and in particular, IL-1 and IL-17 can enhance the MSC osteogenesis as shown in previous in vitro studies (2,3). However, little is known about the role of these cytokine-MSC interactions in the bone-related diseases in humans. Objectives The main focus of this study was to assess if the immune-dependent licensing process of MSCs could be involved in defective bone regeneration. Methods We used samples of bone marrow aspirates (n=15) from two groups of traumatic bone fracture patients; normal union and non-union. Bone marrow MSCs were analyzed for the surface expression of the receptors of the pro-inflammatory licensing cytokines using flowcytometry-optimized panels. Additionally, a comparison of the cytokine effect on the proliferation of cultured MSCs was compared between normal union and non-union groups using the cell proliferation XTT test. Results Interestingly, there were significant lower expression levels of IL-1 receptors 1 and 3 (IL-1R1 and IL-1R3) on non-union MSCs compared to normal-union MSCs (p=0.0478 and p=0.0113 respectively). Furthermore, the surface levels of TNF-αR1 (CD120a) were significantly lesser on non-union MSCs (p=0.0119). There was a clear trend of reduced expression of IL-17 receptors (CD217) on the surface of non-union MSCs, but it was not statistically significant compared to normal-union (p=0.0726). The XTT data showed a significant less proliferation index for IL-1-treated non-union MSCs compared to normal-union MSCs (p=0.0446). Also, a consistent trend of lower proliferation index of non-union MSCs was detected when these cells were treated by IFN-γ, TNF-α or IL-17. Conclusions Together, the lower levels of the pro-inflammatory cytokine receptors indicated a possible mechanism for a defective response of non-union MSCs to the inflammatory signals (particularly IL-1). Further understating of the impact of immune-MSC interactions on human bone healing and regeneration will help to develop new therapies for musculoskeletal diseases involving osteolytic lesions. References Mesenchymal stromal cell 9licensing9: a multistep process. Krampera M. Leukemia. 2011 Sep;25(9):1408–14. Interleukin-1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt-5a/receptor tyrosine kinase-like orphan receptor 2 pathways. Sonomoto K, Yamaoka K, Oshita K, Fukuyo S, Zhang X, Nakano K, Okada Y, Tanaka Y. Arthritis Rheum. 2012 Oct;64(10):3355–63. Proinflammatory T cells and IL-17 stimulate osteoblast differentiation. Croes M, Oner FC, van Neerven D, Sabir E, Kruyt MC, Blokhuis TJ, Dhert WJ, Alblas J. Bone. 2016 Mar; 84:262–70. Acknowledgements This project (no. S-16–132E) was supported by the AO Foundation. Disclosure of Interest None declared

Background and Objectives: Stem cell technology offers a new hope for many chronic disorders patients. The types of stem cells are different with many differences existing between each type. Mesenchymal stem cells (MSCs) represent one type of adult stem cells that can be easily isolated, then re-transplanted to the patients. This offers potential for their future application in treating many disorders without fear of rejection possibility. MSCs can be isolated from different sources e.g. bone marrow (BMSCs) and adipose tissue (ADSCs). In the present study we compared BMSCs and ADSCs isolated from Sprague-Dawley rats. Methods and Results: For this comparison, immunophenotyping, the analysis of growth rates, proliferation by colony forming unit-fibroblast assay, population doubling time, and trilineage differentiation assays were performed for both BMSCs and ADSCs. The findings revealed that despite no difference in immunphenotypic character between BMSC and ADSC, a better proliferative capacity was observed for ADSCs which would advocate their better use in regenerative applications. On the other hand, BMSCs showed more potential for osteogenic and chondrogenic differentiation. Conclusions: Our study showed that, despite many similarities between both types of cells, there are differences existing which can offer assistance on choosing type of cell to be used in specific diseases. Although ADSCs seem more promising for regenerative application generally, BMSCs may represent a better choice for treating bone disorders.

The vast majority of literature pertaining to mesenchymal stem cells (MSC) immunomodulation has focussed on bone marrow-derived MSC that are systemically infused to alleviate inflammatory conditions. Rheumatoid arthritis (RA) is the commonest autoimmune joint disease that has witnessed significant therapeutic advances in the past decade, but remains stubbornly difficult to treat in a subset of cases. Pre-clinical research has demonstrated that bone marrow, adipose, synovial and umbilical cord-derived MSC all suppress the functions of different immune cells thus raising the possibility of new therapies for autoimmune diseases including RA. Indeed, preliminary evidence for MSC efficacy has been reported in some cases of RA and systemic lupus erythromatosis. The potential use of bone marrow-MSC (BM-MSC) for RA therapy is emerging but the use of synovial MSC (S-MSC) to suppress the exaggerated immune response within the inflamed joints remains rudimentary. Synovial fibroblasts that are likely derived from S-MSCs, also give rise to a cell-cultured progeny termed fibroblast-like synoviocytes (FLS), which are key players in the perpetuation of joint inflammation and destruction. A better understanding of the link between these cells and their biology could be a key to developing novel MSC-based strategies for therapy. The review briefly focuses on BM-MSC and gives particular attention to joint niche synovial MSC and FLS with respect to immunoregulatory potential therapy roles.

Bone reconstruction requires the use of autografts from patients’ iliac crest (IC); for large-volume defects bone void fillers and autologous mesenchymal stem cells (MSCs) are often added. The Reamer/Irrigator/Aspirator (RIA) device provides the means of harvesting large amounts of autograft and additionally yields a waste bag containing MSCs, which is currently discarded. The aim of this study was to enumerate and characterise native MSCs from RIA waste bag and compare them to ‘gold-standard’ donor-matched MSCs from IC bone marrow (BM). IC-BM from age matched trauma patients was used as control. In RIA waste bags the median MSC yield established using a colony-forming fibroblast assay was 314333 (range 5 x 104-1.4 x 106), equivalent to approximately one litre of IC-BM aspirate. CD271+ cells were present at high levels in RIA waste bags, had MSC surface phenotype (CD90+CD73+CD105+CD34>sup>-CD61-CD19-CD31-CD33-) and expressed genes associated with multipotentiality, osteogenesis, adipogenesis and angiogenic support. RIA- CD271+ MSCs were transcriptionally similar to donor-matched IC-CD271+ MSCs (76 % transcripts); with the majority of bone-related and Wnt pathway molecules being expressed at comparable levels. Lower-level expression of MCAM/CD146 and 5/13 hypoxia-related molecules was found in RIA-CD271+ MSCs, potentially reflecting their native residence in a more hypoxic environment of the endosteum and bone cortex. These data suggest that long bones contain very large numbers of MSCs, transcriptionally-similar to IC-BM MSCs; they can be procured by reaming using the RIA device and used, following concentration, as autologous and potentially allogeneic bone repair therapy.

Objective Surgically induced periosteal membrane holds great potential for the treatment of large bone defects representing a simple alternative to combinations of exogenous stem cells, scaffolds and growth factors. The purpose of this study was to explore the biological basis for this novel regenerative medicine strategy in man. Methods Eight patients with critical size defects were treated with the induced membrane (IM) technique. After membrane formation 1 cm2 biopsy was taken together with matched, healthy diaphyseal periosteum (P) for comparative analysis. Morphological characteristics, cell composition and growth factor expression were compared. Functional and molecular evaluation of mesenchymal stromal cell (MSC) activity was performed. Results Both tissues shared similar morphology although IM was significantly thicker than P (p = 0.032). The frequency of lymphocytes, pericytes (CD45−CD34−CD146+) and cells expressing markers consistent with bone marrow MSCs (CD45−/lowCD271bright) were 31. 3 and 15.5-fold higher respectively in IM (all p = 0.043). IM contained 3-fold more cells per gramme of tissue with a similar proportion of endothelial cells (CD45−CD31+). Expressed bone morphogenic protein 2, vascular endothelial growth factor and stromal derived factor 1 (SDF-1) are key tissue regeneration mediators. Adherent expanded cells from both tissues had molecular profiles similar to bone marrow MSCs but cells from IM expressed greater than 2 fold relative abundance of SDF-1transcript compared to P (p = 0.043). Conclusion The IM is a thick, vascularised structure that resembles periosteum with a cellular composition and molecular profile facilitating large defect repair and therefore may be described as an “induced-periosteum”. This tissue offers a powerful example of in situ tissue engineering.