Purpose. Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2. Method. Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses. Results. The mean number of cell colonies was 1.25-fold higher after hypoxia culture relative to normoxia. There were no differences in colony diameters. A panel of common protein signatures (CD29, CD90, CD105 and CD151) for stem cells declined in expression after expansion in hypoxia. However, other signatures (CD13, CD34 and CD44) expression level increased under hypoxia, whilst CD73 expression was unchanged. Pellets from hypoxia-expanded MSCs showed on average a 1.4-fold increase in chondrogenic capacity as judged by glycosaminoglycan (GAG) matrix per DNA content relative to normoxia pellets. The gene expression of collagen II, SOX9, aggrecan and matrillin-3 increased by 1.2-, 2-, 1.3- and 1.5-fold, respectively, in pellets formed from hypoxia-expanded stem cells relative to their normoxia counterparts. Conclusion. Expansion of stem cells under hypoxia potentiates their capacity to form cartilage with improved cartilage properties. However, there is a need for signatures to identify stem cells with propensity to form cartilage

Mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and other mesenchymal tissues but are also important modulators of innate and adaptive immune responses. We have capitalized on these important functions to mitigate adverse responses when bone is exposed to pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or prolonged pro-inflammatory cytokines. Our goal was to optimize osteogenesis and mitigate persistent undesired inflammation by: 1. preconditioning MSCs by short term exposure to lipopolysaccharide (LPS) and Tumor Necrosis Factor alpha (TNF-α), 2. genetic modification of MSCs to overexpress Interleukin 4 (IL-4) either constitutively, or as NFκB-responsive IL-4 over-expression cells, and 3. training the MSCs (innate immune memory) by repeated stimulation with LPS. In the first experiment, bone marrow MSCs and macrophages were isolated from femurs and tibias of C57BL/6 mice. MSCs (1×104 cells) were seeded in 24-well transwell plates in the bottom chamber with MSC growth medium. MSCs were treated with 20 ng/ml TNF-α and 1–20 μg/ml LPS for three days. Primary macrophages (2 × 103 cells) were seeded to the insert of a separate transwell plate and polarized into the M1 phenotype. At day four, MSCs and macrophages were washed and the inserts with M1 macrophages were moved to the plates containing preconditioned MSCs at the bottom of the well. Co-culture was carried out in MSC growth medium for 24h. In the second experiment, bone marrow derived macrophages and MSCs were isolated from femora and tibiae of Balb/c male mice. 5×104 macrophages and 1×104 MSCs were seeded in the bottom well of the 24-well transwell plate. The upper chambers were seeded with unmodified MSCs, MSCs preconditioned with 20 ng/ml TNF-α and 20 mg/ml LPS for 3 days, NFκB-IL4 secreting MSCs (all 5×104 cells), or controls without MSCs. Co-culture was carried out in mixed osteogenic-macrophage media with clinically relevant polyethylene or titanium alloy particles. In the third experiment, bone marrow MSCs and macrophages were collected from femurs and tibias of C57BL/6 male mice. The MSCs were stimulated by LPS, washed out for five days, and re-stimulated by LPS in co-culture with macrophages. First, preconditioned MSCs enhanced anti-inflammatory M2 macrophage (Arginase 1 and CD206) expression, decreased pro-inflammatory M1 macrophage (TNF-α/IL-1Ra ratio) expression, and increased osteogenic markers (alkaline phosphatase expression and matrix mineralization) in co-culture. Second, NFκB-IL4 secreting MSCs decreased pro-inflammatory M1 (TNF-α), increased anti-inflammatory M2 (Arg1, IL-1ra) expression, and enhanced the expression of osteogenic factors Runx2 and alkaline phosphatase, in the presence of particles, compared to other groups. Third, LPS-trained MSCs increased anti-inflammatory (Arginase1 and CD206), and decreased the proinflammatory (TNF-α, IL1b, iNOS, and IL6) marker expression in MSC/macrophage co-culture. Transforming MSCs via the techniques of preconditioning, genetic modification, or training (innate immune memory) can modulate/convert a potentially injurious microenvironment to an anti-inflammatory pro-reconstructive milieu. These effects are highly relevant for bone healing in the presence of adverse stimuli. These concepts using transformed MSCs could also be extended to other organ systems subjected to potentially damaging agents

Osteoarthritis (OA) is the fastest growing global health problem, with a total joint replacement being the only effective treatment for patients with end stage OA. Many groups are examining the use of bone marrow or adipose derived mesenchymal stem cells (MSCs) to repair cartilage, or modulate inflammation to promote healing, however, little efficacy in promoting cartilage repair, or reducing patient symptoms over temporary treatments such as micro-fracture has been observed. There is a growing body of literature demonstrating that MSCs derived from the synovial lining of the joint are superior in terms of chondrogenic differentiation and while improvements in clinical outcome measures have been observed with synovial MSCs, results from clinical studies are still highly variable. Based on our results, we believe this variability in clinical studies with MSCs results in part from the isolation, expansion and re-injection of distinct MSCs subtypes in normal vs. OA tissues, each with differing regenerating potential. However, it remains unknown if this heterogeneity is natural (e.g. multiple MSC subtypes present) or if MSCs are influenced by factors in vivo (disease state/stage). Therefore, in this study, we undertook an ‘omics’ screening approach on MSCs from normal and OA knee synovial tissue. Specifically, we characterized their global proteome and genomic expression patterns to determine if multiple MSC from normal and OA joints are distinct at the protein/gene expression level and/if so, what proteins/genes are differentially expressed between MSCs derived from normal and OA synovial tissue. Synovium tissue was collected from OA patients undergoing joint replacement and normal cadaveric knees. The in vitro adipogenic, chondrogenic and osteogenic differentiation potential of the MSCs was analyzed via qPCR and histology. Fully characterized MSC populations where then analyzed through an unbiased shotgun proteomics, and microarray analysis. Synovial MSCs isolated from both OA and normal knees demonstrated similar multipotent differentiation capacity. Likewise, both OA and normal MSCs display the typical MSCs cell surface marker profile in vitro (CD90+, CD44+, CD73+, CD105+). Using shotgun proteomics, 7720 unique peptides corresponding to 2183 proteins were identified and quantified between normal and OA MSCs. Of these 2183 proteins, 994 were equally expressed in normal and OA, MSCs, 324 were upregulated in OA MSCs (with 50 proteins exclusively expressed in OA MSCs), 630 proteins were upregulated in normal MSCs (with 16 proteins exclusively expressed in normal MSCs). Microarray analysis of normal and OA MSCs demonstrated a similar result in where, 967 genes were differentially expressed between normal and OA MSCs, with 423 genes upregulated in OA, and 544 genes upregulated in normal MSCs. In this project, we have demonstrated that although normal and OA synovial derived MSCs demonstrate similar multipotent differentiation potential and cell surface markers expression, these cells demonstrated significant differences at the molecular level (protein and gene expression). Further research is required to determine if these differences influence functional differences in vitro and/or in vivo and what drives this dramatic change in the regulatory pathways within normal vs. OA synovial MSCs

Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells. DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of mouse embryos and in adult human cartilage obtained from knee replacement. RNAi of DACT1 and DACT2 was performed on isolated chondrocytes and MSCs from human bone marrow. Knockdown efficiency and cell morphology was confirmed by qPCR and immunofluorescence. To understand which pathways are affected by DACT1, we performed next-generation sequencing gene expression analysis (RNAseq) on cells where DACT1 had been reduced by RNAi. Top statistically significant (p < 0 .05) 200 up and downregulated genes were analysed with Ingenuity® Pathway Analysis software. We observed DACT1 and DACT2 in chondrocytes throughout the osteoarthritic tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 was localised to the articular surface. Furthermore, in mouse embryos (E.15.5), we observed DACT2 at the interzones, sites of developing synovial joints, suggesting that DACT2 has a role in cartilage progenitor cells. We subsequently analysed the expression of DACT1 and DACT2 in MSCs and found that both are expressed in synovial and bone marrow-derived MSCs. We then performed an RNAi knockdown experiment. DACT1 knockdown in both chondrocyte and MSCs caused the cells to undergo apoptosis within 24 hours. The RNA-seq study of DACT1 silenced bone marrow-derived MSCs, from 4 different human subjects, showed that loss of DACT1 has an effect on the expression of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways. In summary, we describe for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. Loss of DACT1 induced cell death in both chondrocytes and MSCs, with RNA-seq analysis revealing a direct impact on transcript levels of genes involved in the Wnt and TFGβ signalling, key regulatory pathways in skeletal development and repair

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function. Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs

The ability of mesenchymal stem cells (MSCs) to differentiate in vitro into chondrocytes, osteocytes and myocytes holds great promise for tissue engineering. Skeletal defects are emerging as key targets for treatment using MSCs due to the high responsiveness of bone to interventions in animal models. Interest in MSCs has further expanded in recognition of their ability to release growth factors and to adjust immune responses. Despite their increasing application in clinical trials, the origin and role of MSCs in the development, repair and regeneration of organs have remained unclear. Until recently, MSCs could only be isolated in a process that requires culture in a laboratory; these cells were being used for tissue engineering without understanding their native location and function. MSCs isolated in this indirect way have been used in clinical trials and remain the reference standard cellular substrate for musculoskeletal engineering. The therapeutic use of autologous MSCs is currently limited by the need for ex vivo expansion and by heterogeneity within MSC preparations. The recent discovery that the walls of blood vessels harbour native precursors of MSCs has led to their prospective identification and isolation. MSCs may therefore now be purified from dispensable tissues such as lipo-aspirate and returned for clinical use in sufficient quantity, negating the requirement for ex vivo expansion and a second surgical procedure. In this annotation we provide an update on the recent developments in the understanding of the identity of MSCs within tissues and outline how this may affect their use in orthopaedic surgery in the future. Cite this article: Bone Joint J 2014;96-B:291–8

Osteoarthritis (OA) is one of the most prevalent joint diseases involving progressive and degenerative changes to cartilage resulting from a variety of etiologies including post-traumatic incident or aging. OA lesions can be treated at its early stages through cell-based tissue engineering therapies using Mesenchymal Stem Cells (MSCs). In vivo models for evaluating these strategies, have described both chondral (impaction) and osteochondral (biopsy punch) defects. The aim of the investigation was to develop a compact and reproducible defect inducing post-traumatic degenerative changes mimicking early OA. Additionally, a pilot study to evaluate the efficacy of MSC-hydrogel treatment was also assessed. Surgery was performed on New Zealand white rabbits (male, 5–8 months old) with defects created on medial femoral condyle. For developing an appropriate defect, three approaches were used for evaluation: a biopsy punch (n = three at six and twelve weeks), an impaction device1 (n = three at six and twelve weeks) and a dental drill model (n = six at six and twelve weeks). At stated time points, condyles were harvested and decalcified in 10% EDTA, then embedded in Tissue-Tek and sectioned using a cryostat. Upon identification of region of interest, sections were stained with Safranin-O/Fast green and scored using OARSI scoring system by two blinded observers2. For the pilot study, autologous bone marrow was harvested from rabbits and used to isolate and expand MSCs. The Dental drill model was applied to both knee condyles, left untreated for six weeks at which stage, PKH26 fluorescently labelled MSCs were seeded into a hyaluronic acid hydrogel (TETEC). Repair tissue was removed from both condyles and MSC-hydrogel was injected into the left knee, whilst right knee was left empty. Rabbits were sacrificed at one (n = 1), six (n = 3) and twelve (n = 3) weeks post-treatment, processed as previously described and cartilage regeneration evaluated using Sellers score3. Impacted condyles exhibited no observed changes histologically (Mean OARSI score = 1 + 1), whereas biopsy punched and dental drilled defects demonstrated equal signs of cartilage erosion (OARSI score = 3 + 1) at assessed time points. However, biopsy punched condyles formed a diffusive defect, whereas dental drilled condyles showed a more defined, compact and reproducible defect. In the pilot study, PKH-labelled MSCs were observed at one and six weeks post-implantation within the defect space where hydrogel was injected. Tissue regeneration assessment indicated no difference between empty (Mean Sellers score = 14 + 2) and MSC treated defects (Sellers score = 16 + 5) at six weeks post-injection. At twelve weeks, MSC treated defects showed improved tissue regeneration with substantial subchondral bone restoration and good integration of regenerative cartilage with surrounding intact tissue (Sellers score = 10 + 1), whereas untreated defects showed no change in regeneration compared to six weeks (Sellers score = 16 + 2). Dental drill model was found to be the appropriate strategy for investigating early OA progression and treatment. Application of MSCs in defects showed good cartilage regeneration after twelve weeks application, indicating their promise in the treatment of early OA defects

Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a major cause of disability in the adult population with its prevalence expected to increase dramatically over the next 20 years. Although current therapies can alleviate symptoms and improve function in early course of the disease, OA inevitably progresses to end-stage disease requiring total joint arthroplasty. Mesenchymal stromal cells (MSCs) have emerged as a candidate cell type with great potential for intra-articular (IA) repair therapy. However, there is still a considerable lack of knowledge concerning their behaviour, biology and therapeutic effects. To start addressing this, we explored the secretory profile of bone marrow derived MSCs in early and end-stage knee OA synovial fluid (SF). Subjects were recruited and categorised into early [Kellgren-Lawrence (KL) grade I and II, n=12] and end-stage (KL grade III and IV, n=11) knee OA groups. The SF proteome of early and end-stage OA was tested before and three days after the addition of bone marrow MSCs (16.5×10^3, single donor) using multiplex ELISA (64 cytokines) and mass spectrometry (302 proteins detected). Non parametric Wilcoxon-signed rank test for paired samples was used to compare the levels of proteins before and after addition of MSCs in early and end-stage knee OA SF. Significant differences were determined after multiple comparisons correction (FDR) with a p<0.05. Gender distribution and BMI were not statistically different between the two cohorts (p>0.05). However, patients in early knee OA cohort were significantly younger (44.7 years, SD=7.1) than patients in the end-stage cohort (58.6 years, SD=4.4; p<0.05). In both early and end-stage knee OA, MSCs increased the levels of VEGF-A (by 320.24 pg/mL), IL-6 (by 826.78 pg/mL) and IL-8 (by 128.85 pg/mL), factors involved in angiogenesis; CXCL1/2/3 (by 103.35 pg/mL), CCL2 (by 1187.27 pg/mL), CCL3 (by 15.82 pg/mL) and CCL7 (by 10.43 pg/mL), growth factors and chemokines. However, CXCL5 (by 48.61 pg/mL) levels increased only in early knee OA, whereas PDGF-AA (by 15.36 pg/mL) and CXCL12 (by 497.19 pg/mL) levels increased only in end-stage knee OA. This study demonstrates that bone marrow derived MSCs secrete angiogenic and chemotactic factors both in early and end-stage knee OA. More importantly, MSCs show a differential reaction between early and end-stage OA. Functional assays are required to further understand on how the therapeutic effect of MSCs is modulated when exposed to OA SF

Purpose. The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro. Method. MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time quantitative PCR on day 14. Also assessed was alkaline phosphatase (ALP) activity by a colorimetric kinetic assay and Fast Blue B staining on day 14. Calcium deposition was determined by the calcium assay on day 35. Results. The expression levels of integrin binding sialoprotein (IBSP) and osteocalcin (OC) were significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT and control groups (all p<0.038). Although the expression level of runt-related transcription factor 2 (RUNX2) was also reduced in NOGsi1 and NOGsi2 groups compared with NT and control groups, it did not reach statistical significance. ALP activity was significantly lower in NOGsi1 and NOGsi2 groups than that of NT group (both p<0.024). The same pattern was also observed in ALP Fast Blue B staining. Calcium deposition was also significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT group (both p<=0.048). Conclusion. Noggin suppression by siRNA inhibits BMP-2-induced osteogenesis of human bone marrow-derived MSCs. Our results, contrary to the extensive studies conducted in rodent cells/models, corroborated with the previous study that the addition of noggin in the cell culture increased osteogenesis of human MSCs. This suggests that the effects of noggin on BMP-2-induced osteogenesis of MSCs might be species-specific

Osteogenesis Imperfecta (OI) is a heritable bone disorder characterized by bone fragility and often caused by mutations in the Type I collagen-encoding genes COL1A1 and COL1A2. The pathophysiology of OI, particularly at the cellular level, is still not well understood. This contributes to the lack of a cure for this disorder as well as an effective preventive or management options of its complications. In the bone environment, mesenchymal stem cells (MSCs) and osteoblasts (Ob) exert their function, at least partially, through the secretion of extracellular vesicles (EV). EV is a heterogeneous group of nanosized membrane-enclosed vesicles that carry/transfer a cargo of proteins, lipid and nucleic acids from the secreting cell to its target cells. Our objective is to characterize EVs secreted by human control (HC)- and OI-MSCs and their derived Obs, with focus on their protein content. We hypothesize that there will be differences in the protein content of EVs secreted by OI-Obs compared to HC-Ob, which may indicate a deviation from healthy Ob behavior and, thus, a role in OI pathophysiology. MSCs were harvested from the adipose tissue of four COL1A1-OI and two HC patients. They were proliferated in an EV-depleted media, then induced to differentiate to extracellular matrix (ECM)-producing osteoblasts, which then gets mineralized. EVs secreted by MSCs (MSC-EV) and Obs (Ob-EV) were then purified and concentrated. Using liquid chromatography- tandem mass spectrometry, proteomic analysis of the EV groups was done. A total of 384 unique proteins were identified in all EVs, 373 were found in Vesiclepedia indicating a good enrichment of our samples with EV proteins. 67 proteins of the total 384 were exclusively or significantly upregulated (p-value < 0 .05) in OI-Ob-EV and 28 proteins in the HC-Ob-EVs, relative to each other. These two groups of differentially expressed proteins were compared by Gene Ontology (GO) analysis of their cellular compartment, molecular functions and biological processes. We observed that there were differences in the cellular origin of EV-proteins, which may indicate heterogeneity of the isolated EVs. Molecular function and biological process analyses of the HC-Ob-EV proteins showed, as expected, predominantly calcium-related activities such as extracellular matrix (ECM) mineralization. OI-Ob-EV proteins were still predominantly exhibiting ECM organization and formation functions. Annexins A1,2,4,5 and 6 were differentially and significantly upregulated by the HC-Ob-EVs. Fibronectin (FN), Fibulin-1 and −2, and Laminins (α4 & γ1), which are amongst the early non-collagenous proteins to form the ECM, were differentially and significantly upregulated in the OI-Ob-EVs. We concluded that the persistent expression of Fibronectin (FN), Fibulin-1 and −2, and Laminins in OI-Ob-EVs might indicate the presence of an immature ECM that the OI-Obs are trying to organize. ECM mineralization is largely dependent on the presence of an organized mature ECM, and this being compromised in OI bone environment, may be a contributor to the bone fragility seen in these patients. Annexins, which are calcium-binders that are vital for ECM mineralization, were significantly downregulated in the OI-Ob-EVs and this may be a further contributor to ECM mineralization impairment and bone fragility

Previous study reported that intra-articular injection of MgSO4 could alleviate pain related behaviors in a collagenase induced OA model in rats. It provided us a good description on the potential of Mg2+ in OA treatment. However, the specific efficiency of Mg2+ on OA needs to be further explored and confirmed. The underlying mechanisms should be elucidated as well. Increasing attention has been paid on existence of synovial fluid MSCs (SF-MSCs) (not culture expanded) which may participate in endogenous reparative capabilities of the joint. On the other hand, previous studies demonstrated that Mg2+ not only promoted the expression of integrins but also enhanced the strength of fibronectin-integrin bonds that indicated the promotive effect of Mg2+ on cell adhesion, moreover, Mg2+ was proved could enhance chondrogenic differentiation of synovial membrane derived MSCs by modulating integrins. Based on these evidence, we hypothesize herein intra-articular injection of Mg2+ can attenuate cartilage degeneration in OA rat through modulating the biological behavior of SF-MSCs. Human and rat SF-MSCs were collected after obtaining Experimental Ethics approval. The biological behaviors of both human and rat SF-MSCs including multiple differentiation, adhesion, colony forming, proliferation, etc. were determined in vitro in presence or absence of Mg2+ (10 mmol/L). Male SD rats (body weight: 450–500 g) were used to establish anterior cruciate ligament transection and partial medial meniscectomy (ACLT+PMM) OA models. The rats received ACLT+PMM were randomly divided into saline (control) group and MgCl2 (0.5 mol/L) group (n=6 per group). Intra-articular injection was performed on week 4 post-operation, twice per week for two weeks. Knee samples were harvested on week 2, 4, 8, 12 and 16 after injection for histological analysis for assessing the progression of OA. On week 2 and 4 after injection, the rat SF-MSCs were also isolated before the rats were sacrificed for assessing the abilities of chondrogenic differentiation, colony forming and adhesion in vitro. Statistical analysis was done using Graphpad Prism 6.01. Unpaired t test was used to compare the difference between groups. Significant difference was determined at P < 0 .05. The adhesion and chondrogenic differentiation ability of both human and rat SF-MSCs were significantly enhanced by Mg2+ (10 mmol/L) supplementation in vitro. However, no significant effects of Mg2+ (10 mmol/L) on the osteogenic and adipogenic differentiation as well as the colony forming and proliferation. In the animal study, histological analysis by Saffranin O and Toluidine Blue indicated the cartilage degeneration was significantly alleviated by intra-articular injection of Mg2+, in addition, the expression of Col2 in cartilage was also increased in MgCl2 group with respect to control group indicated by immunohistochemistry. Moreover, the OARSI scoring was decreased in MgCl2 group as well. Histological analysis and RT-qPCR indicated that the chondrogenic differentiation of SF-MSCs isolated from Mg2+ treated rats were significantly enhanced compare to control group. In the current study, we have provided direct evidence supporting that Mg2+ attenuated the progression of OA. Except for the effect of Mg2+ on preventing cartilage degeneration had been demonstrated in this study, for the first time, we demonstrated the promoting effect of Mg2+ on adhesion and chondrogenic differentiation of endogenous SF-MSCs within knee joint that may favorite cartilage repair. We have confirmed that the anti-osteoarthritic effect of Mg2+ involves the multiple actions which refer to prevent cartilage degeneration plus enhance the adhesion and chondrogenic differentiation of SF-MSCs in knee joint to attenuate the progression of OA. These multiple actions of Mg2+ may be more advantage than traditional products. Besides, this simple, widely available and inexpensive administration of Mg2+ has the potential on reducing the massive heath economic burden of OA. However, the current data just provided a very basic concept, the exact functions and underlying mechanisms of Mg2+ on attenuating OA progression still need to be further explored both in vitro and in vivo. Formula of Mg2+ containing solution also need to be optimized, for example, a sustained and controlled release delivery system need to be developed for improving the long-term efficacy

MSCs (mesenchymal stem cells) are bone marrow-derived cells capable of replication and differentiation in-vitro into several tissues including bone, cartilage, stroma, fat, muscle and tendon. MSCs can be isolated by relatively simple procedures and then expanded without losing the ability to differentiate into multiple lineages. As such, these cells have immense clinical potential in regenerative medicine and in orthopaedics for repair or replacement of damaged tissues. In this work we investigated the interaction between magnetic carbon nanotubes (CNTs) and MSCs and their ability to guide these cells injected intravenously in living mice by using an external magnetic field. CNTs did not affect cell viability and their ability to differentiate. Both the CNTs and the magnetic field did not alter cell growth rate, phenotype and cytoskeletal conformation. CNTs, when exposed to magnetic fields, are able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the biodistribution of CNT-labelled MSCs after intravenous injection into rats. We demonstrated that CNTs hold the potential for use as nano-devices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for manipulation/guidance of MSCs in regenerative medicine and cell transplantation for the treatment of many orthopaedic diseases

INTRODUCTION. The uncertainty of the biological effects of wear and corrosion from Metal-on-metal (MoM) implants has initiated a debate on their safety and use. Generally, the release of wear particles from MoM hip implants can clinically manifest in aseptic osteolysis. In our study, the effect of MoM-wear particles and particle originated Co and Cr ions on mesenchymal stromal cells (MSCs) was investigated [1]. The lead hypotheses were that (1) dissociated Co and Cr, originated from MoM-wear particles, accumulate in the bone marrow and (2) apparently impair the osteogenic function of local MSCs. This impairment could be one element contributing to the manifestation of periprosthetic osteolyses. METHODS. The study was approved by the local ethical committee (EA1/194/13); all donors gave written informed consent. Blood (B), Synovial fluid (SF) periprosthetic tissue (PT) and bone marrow (BM) were collected from patients with at least one osteolytic lesion, undergoing a revision of a MoM hip implant. Patients undergoing primary THA served as controls. Metal wear particles were isolated from PT by enzymatic digestion and their size and shape characterized by transmission electron microscopy (TEM). Local and systemic levels of Co and Cr were analyzed by graphite furnace atomic absorption spectroscopy. MoM-MSCs and control-MSCs were isolated from BM for in vitro assessment of their viability, proliferation, migration and multilineage differentiation. In addition, control-MSCs were in vitro exposed to Co and Cr ions and assessed for their viability, proliferation and osteogenic differentiation. RESULTS. We confirmed the presence of nanoscaled particles that appeared heterogeneous in size and shape (mean diameter: 34.7 ± 16.2 nm; n = 64). Second, we confirmed the exposure to Co and Cr and quantified its particulate and dissociated amounts. Local metal levels exceed systemic ones by several magnitudes and dissociated Co and Cr was found in periprosthetic compartments including BM (peak BM Co concentration: 977 µg/L, peak BM Cr concentration: 2,875 µg/L; n = 10). While in vivo exposure to MoM-wear did not influence MSCs' viability, proliferation, migration capacity, adipogenic- and chondogenic differentiation, it decreased osteogenic matrix mineralization and cellular ALP activity (FIGURE 1). In vitro exposure of control-MSCs to Co(II) and Cr(III) ions, at concentrations detected in BM, confirmed the decrease in osteogenic matrix mineralization and ALP activity (FIGURE 2). DISCUSSION. We found that extensive amounts of Co and Cr occurred in their dissociated state within the periprosthetic region (SF, PT, BM), indicating that vast amounts of particulate wear in degraded. Our work represents an important piece in the puzzle of the clinical manifestation of periprosthetic osteolysis: dissociated Co and Cr at clinically relevant concentrations impair the bone forming function of MSCs. The study's data indicate an influence of MoM-wear on BM residing MSCs' osteogenic differentiation that is relevant for maintaining vital bone structure, thus confirming the lead hypothesis. The use of CoCrMo alloys for articulating surfaces in hip implants needs critical reconsideration. For figures/tables, please contact authors directly.

Purpose. Whilst it is known that oxidative stress can cause early degenerative changes observed in experimental osteoarthritis and that a major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients express type X collagen, a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification), little is known whether the expression of type X collagen in MSCs from OA patients can be related to oxidative stress or inflammatory reactions that occur during this disease. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Bone marrow aspirates were processed essentially as previously described. Briefly, non-adherent cells were discarded after 72h of culture and the adherent ones were expanded for 2–3 passages. MSCs from normal donor (control) were obtained from Lonza. Cells were then lysed and protein expression was detected by Western blot using specific antibodies directed against type X collagen, as well as the antioxidant enzymes Mn-superoxide dismutase (MnSOD), catalase (CAT) and glutathione peroxidase-1 (GPx-1) and inflammation related proteins cyclooxygenase-1 (COX-1) and intercellular adhesion molecule-1 (ICAM-1). GAPDH was used as a housekeeping gene and served to normalize the results. Correlations between the expressions of the different proteins were realized using the correlation Z test with StatView (SAS Institute). Results. Results confirmed that type X collagen was over-expressed in MSCs from OA patients when compared to expression in cells of normal donors. MnSOD, CAT, and COX-1 were also over-expressed. Results showed that the expression of MnSOD strongly correlated to the expression of type X collagen (r=0.79; p=0.03). The expression of CAT weakly correlated to the expression of type X collagen (r=0.67; p=0.10) whereas GPx was not expressed in MSCs from OA patients. Regarding inflammatory reaction, results showed that COX-1 expression strongly correlated to type X collagen expression (r=0.77; p=0.004). ICAM-1 was weakly expressed and no correlation with the expression of type X collagen was observed. Interestingly, COX-1 expression was highly correlated to the expression MnSOD (r=0.92; p=0.0001) and the expression of CAT (r=−0.82; p=0.02). Conclusion. We showed that the level of anti-oxidant enzymes correlates with type X collagen expression in MSCs from OA patients. This suggests that oxidative stress may lead to the up-regulation of stem cell hypertrophy. Results also suggest that prostaglandin production though COX-1 activity is associated with anti-oxidant enzyme expression (MnSOD) and hypertrophy (type X collagen expression). Further studies are however necessary to better understand whether the increased expression of these proteins is the cause or the effect of type X collagen over-expression in MSCs from OA patients

Purpose. Mesenchymal stromal cells (MSCs) are an attractive choice for regenerative medicine. We previously showed that MSCs enhance wound healing in animals after radiotherapy. The effect of MSCs on tumor growth is not well understood. The potential use of MSCs to enhance wound healing after radiotherapy (RT) and resection of soft tissue sarcoma (STS) is dependent on a satisfactory safety profile to ensure that tumor proliferation does not occur and recurrence is not increased. Method. Primary cell lines (human myxofibrosarcoma and undifferentiated sarcoma) derived from sarcoma bearing patients and a commercialized human fibrosarcoma cell line (HT1080) were used. Cell line proliferation assay after co-culture with MSCs was done using flow cytometry (CFSE) and bioluminescence emission (BLI) (using eGFP/Fluc transduced cell lines). Five xenograft models were developed with NOD/SCID gc-null mice (n=164) harbouring primary tissue lines obtained from patients biopsies (myxofibrosarcoma and three pleomorphic undifferentiated sarcoma [PUS A, B and C]) and a a fibrosarcoma cell line previously transduced with eGFP/Fluc. Tumors were passaged to three mouse generations before a tissue line was established and the model was then used. For the fibrosarcoma model, eGFP/Fluc HT1080 were injected under the dorsal skin. When tumors reached 1cm in diameter, they received localized RT and 48hr later were resected. MSCs (n=82) or medium alone (n=82) was injected subcutaneously adjacent to the wound after tumor resection. Histological and in vivo BLI analysis were performed 3 and 12 weeks after surgery. Results. In Vitro Proliferation Assay. For the flow cytometric proliferation assay, there was an increase in the doubling time after five days in the myxofibrosarcoma-MSCs co-culture system (140.4h) compared with controls (55.4 h, p<0.001). No significant differences were found in other cells lines. Lower BLI emission was found in co-cultured myxofibrosarcoma cells at the 3rd and 4th day compared with controls (p<0.01 and p<0.05 respectively). In Vivo Recurrence Assay. For mice bearing the fibrosarcoma cell line, in vivo BLI performed 3 weeks after surgery showed similar emission intensity in MSC-treated mice and controls while histological recurrence was significantly lower in MSC-treated animals (40%) than control (72%, p=0.045). For mice bearing the myxofibrosarcoma tissue line, histological recurrence at 12 weeks was similar in MSCs-treated animals and controls (p=0.44). Mice xenografted with pleomorphic undifferentiated sarcoma A and B did not develop local tumor recurrence after histological analysis, while pleomorphic undifferentiated sarcoma C showed similar recurrence in MSC and medium treated mice (p=0.46). Conclusion. We showed that MSCs decrease the proliferation rate of the myxofibrosarcoma cell line in vitro and have no effect, or even decrease, local recurrence of different STS tissue lines in vivo after RT and resection. Clinical investigation of this approach is warranted

Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

Bone marrow concentrates are being used to augment soft tissue healing. However, only 0.01% of these cells meet the criteria of a mesenchymal stem cell (MSC), which likely accounts for the variability in reported results. Previous studies using an established rat rotator cuff repair model have demonstrated that bone marrow-derived MSCs had no effect on healing. In this study we evaluated the effect of purified human MSCs on rotator cuff healing in an athymic rat model. Hypothesis: Purified human MSCs added to the repair site will improve biomechanical strength and fibrocartilage formation of the healing tendon. Fifty-two athymic rats underwent unilateral detachment and repair of the supraspinatus tendon with either fibrin glue (control) or fibrin glue with 106 hMSCs (experimental) applied at the repair site. Flow cytometry verified the stem cell phenotype of the cells as CD73+, CD90+, CD105+, CD14-, CD34- and CD45-. Rats were sacrificed at 2 and 4 weeks, with 10 used for biomechanical testing and 3 for histologic analysis from each group. Biomechanical testing revealed a significant increase in failure load (11.5±2.4N vs. 8.5±2.4N, p=0.002) and stiffness (7.1±1.2 N/mm vs. 5.7±2.1 N/mm, p0.17). These data demonstrate the potential for stem cells to augment tendon healing. This is the first study to use purified stem cells, rather than simple bone marrow concentrate. In the future, cell sorting techniques and culture expansion could be used to select and expand the small population of true stem cells in bone marrow. Furthermore, healing could potentially be improved with repeat cell injection at an additional post-operative time point

The meniscus is at the cornerstone of knee joint function, imparting stability and ensuring shock absorption, load transmission, and stress distribution within the knee joint. However, it is very vulnerable to injury and age-related degeneration. Meniscal tears are reported as the most common pathology of the knee with a mean annual incidence of 66 per 100,000. Knee osteoarthritis progresses more rapidly in the absence of a functional meniscus. Historically, tears extending to the avascular inner portion of the meniscus (white-white zone, “WW”), such as radial tears were considered as untreatable and were often resected, due to the lack of vascularity in the WW zone. Perfusion-based anatomical studies performed on cadaveric menisci in the 1980s shaped the current dogma that human meniscus has poor regenerative capacity, partly due to limited blood supply that only reaches 10 to 25% of the meniscus, commonly referred to as red-red zone (“RR”). Previous studies, including those utilizing animal models have shown mobilization of Mesenchymal Stem Cells (MSCs) upon injury into the WW zone, and successful MSC recruitment when administered externally to the injury site. We and others have recently reported positive outcomes of repaired tears in the inner zone of patients. We hypothesized that the “avascular” white-white zone of the meniscus possesses regenerative capacity due to a resident stem/progenitor cell population. Further, we sought to redefine the presence of microvessels in all meniscal zones using advanced stereology and imaging modalities. Fifteen menisci from fresh human cadaveric knees (mean age: 21.53±6.53 years) without evidence of previous injury were obtained from two tissue banks (JRF, Centennial, CO) and Biosource Medical (Lakeland, FL) and utilized for this study. The use of cadaveric specimens for research purposes was approved by the institutional review board. Tibial plateaus were dissected to harvest medial and lateral menisci along their entire length. The RR, red-white (RW) and WW zones were dissected and separated into three thirds from the inner aspect to the marginal border of the meniscus and their wet weights recorded (Fig.1A). Meniscus tissue cellular content in each zone was obtained from dissociation of meniscus tissue using 0.02% w/v pronase (Millipore) for 1h at 37oC, followed by 18h 0.02% w/v collagenase II (Worthington) at 37oC with shaking. Isolated cells were characterized immediately after harvest using flow cytometry with antibodies against MSCs surface markers (CD105, CD90, CD44 and CD29) as well as respective isotype controls. Further, meniscal cells were cultured and split twice when confluence was reached, characterized at P2 and compared to bone marrow-derived MSCs (BM-MSCs) using the same markers. Self-renewal of cells was assessed using colony forming unit (CFU) assay. Differentiation assays were performed to assess whether colony-forming cells retained multilineage potential. For morphological examination of bigger vessels, samples were fixed in 10% formalin for 1 week, paraffin embedded, sectioned (4 μm thick) and stained with H&E and Masson's trichrome. Presence of microvessels was assessed by CD31 immunofluorescence staining. Further, menisci were cleared using the uDisco protocol labeled with the TO-PRO®-3 stain, a fluorescent dye that stains cell nuclei and imaged using light-sheet microscopy. All continuous data are presented as mean ±standard deviation. Non-repeated measures analysis of variance (ANOVA) and Tukey-Kramer HSD post hoc analysis were performed on sample means for continuous variables. Statistical significance was set at p < 0 .05. Menisci were successfully cleared using a modified uDISCO procedure, imaged and analyzed for total cell density. As expected, bigger vessels were observed in RR but not in WW. However, immunofluorescent staining for CD31 showed a subset of CD31+endothelial cells present in the WW zone, indicating the presence of small vessels, most likely capillaries. In order to assess whether enzymatic digestion had a differential result depending on meniscus zone due to cellular content, we analyzed yields per meniscus per zone. The wet weight of different zones (WW:RW:RR) was at a ratio of ∼1:3:5 respectively, however, the ratio of cells isolated from each zone was at ∼1:4:20, indicating that RR has a denser population of mononuclear cells. However, the difference between all zones in cell yields was not significant. The clonogenic potential of isolated cells was shown to be non-significantly different between the three zones. Differentiation of isolated cells to osteogenic lineage using osteogenic media in vitroshowed no difference between the three zones. Flow cytometry analysis of cells from the three meniscal zones displayed presence of two distinct subpopulations of cells immediately after isolation. One subpopulation was positive to MSC surface markers and the other negative. Additionally, flow cytometry of cultured meniscal cells at P2 displayed that the entire cell population was CD44+CD105+CD29+CD90+, suggesting that culturing meniscal cells results in selection of stem/progenitor cells (plastic adherence). Surface marker expression analysis showed differential expression patterns between markers depending on zone. Similar fraction of cells was detected to express both MSC markers CD90 and CD105 (7–10%) and similar fraction of cells expressed both MSC markers CD29 and CD44 (1–2%) in all three zones, indicating similar density of resident stem/progenitor cells in each zone. Importantly, WW showed significantly higher expression for all four MSC markers compared to the RR zone, indicating higher relative density of stem/progenitor resident cells in the WW zone. Our results determine that CD31-expressing microvessels were present in all zones, including the WW zone, which was previously considered completely avascular. Additionally, stem/progenitor cells were shown to be present in all three zones of the menisci, including the WW zone, showcasing its regenerative potential. For any figures or tables, please contact the authors directly

Background. 70% of breast cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self-renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential. Aim. To investigate interactions between MSCs and breast cancer cells to further elucidate their role in the tumour microenvironment and hence understand factors involved in stimulating the formation of bone metastases. Methods. MSCs harvested from the iliac crest of healthy volunteers were grown for collection of conditioned medium (CM), containing all factors secreted by the cells. Breast cancer cell lines (T47D, SK-BR-3, MDA-MB-231) were then cultured in MSC CM +/− antibodies to TGFβ, VEGF, MCP-1 and CCL5 for 72hrs. Cell proliferation was assessed using an Apoglow. (r). assay and RNA harvested for analysis of changes in Epithelial Mesenchymal Transition specific gene expression : N-Cadherin, E-Cadherin, Vimentin, Twist, Snail. Results. A significant down regulation of breast cancer cell proliferation in the presence of MSC secreted factors was observed (p< 0.05). There was a dramatic increase in expression of EMT specific genes in both cell lines following exposure to MSC-secreted factors. Inclusion of antibodies to TGF, VEGF, MCP-1 and CCL5 inhibited the effect seen, suggesting these paracrine factors played a role in the elevated expression levels. Conclusion. MSCs clearly have a distinct paracrine effect on breast cancer epithelial cells, mediated at least in part through secretion of growth factors and chemokines. These factors play an important role in the metastatic cascade and may represent potential therapeutic targets to inhibit MSC-breast cancer interactions, helping to prevent the formation of bone metastases in cancer

Background. 70% of Breast Cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential. Aims. The aim of this study was to investigate interactions between MSCs and breast cancer cells to further elucidate their role in the tumour microenvironment and hence understand factors involved in stimulating the formation of bone metastases. Methods. MSCs harvested from the iliac crest of healthy volunteers were grown for collection of conditioned medium (CM), containing all factors secreted by the cells. Breast cancer cell lines (T47D, SK-BR-3, MDA-MB-231) were then cultured in MSC CM +/− antibodies to TGFβ, VEGF, MCP-1 and CCL5 for 72hrs. Cell proliferation was assessed using an Apoglow(r) assay and RNA harvested for analysis of changes in Epithelial Mesenchymal Transition specific gene expression : N-Cadherin, E-Cadherin, Vimentin, Twist, Snail. Results. A significant down regulation of breast cancer cell proliferation in the presence of MSC secreted factors was observed (p<0.05). There was a dramatic increase in expression of EMT specific genes in both cell lines following exposure to MSC-secreted factors. Inclusion of antibodies to TGF, VEGF, MCP-1 and CCL5 inhibited the effect seen, suggesting these paracrine factors played a role in the elevated expression levels. Conclusion. MSCs clearly have a distinct paracrine effect on breast cancer epithelial cells, mediated at least in part through secretion of growth factors and chemokines. These factors play an important role in the metastatic cascade and may represent potential therapeutic targets to inhibit MSC-breast cancer interactions, helping to prevent the formation of bone metastases in cancer