The enthesis is a tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Given the avascularity of the tendon and the gradual change in tissue architecture and cell phenotype, the enthesis original tissue is often not re-established after chronic injuries, resulting in scar formation. Conservative treatments and surgical approaches are still far from a functional regeneration, whilst tissue engineering based scaffolds have recently showed great potential. In this work, we hypothesised that collagen-based scaffolds that mimic the basic architecture of the enthesis, will be able to spatially direct stem cell differentiation, providing an in vitro platform to study enthesis regeneration. A three-layer sponge composed of a tendon-like layer (collagen type I), a fibrocartilage-like layer (collagen type II) and a bone-like layer (collagen type I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold pore size and structural continuity at the interfaces were assessed by SEM and μ-CT analysis. Bone-marrow derived stem cells (BMSCs) were seeded on the scaffold and cultured in basal and differentiation media (chondrogenic, tenogenic and osteogenic). At day 7 and 21 the scaffolds were stained with Alizarin Red and Alcian Blue; alkaline phosphatase activity (ALP) and calcium and glycosaminoglycans (GAGs) were quantified in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I, III, tenascin and decorin in the scaffolds was evaluated by immunofluorescence staining in order to evaluate tenogenic differentiation of BMSCs. Scaffolds with three distinct but interconnected layers of collagen type I, collagen type II and collagen type I + hydroxyapatite were fabricated, with pore sizes in the range of 100–200 μm. Increased ALP and calcium levels were detected in a localised manner within the bone-like layer when scaffolds were cultured in basal medium (p<0.025 vs the other 2 layers). Similarly, proteoglycans were detected specifically in the fibrocartilage-like layer when scaffolds were cultured in the chondrogenic differentiation medium (p<0.03 vs the other 2 layers). Increased expression of tenogenic markers was observed in the tendon-like layer of scaffolds cultured in tenogenic media (p<0.045 vs the other 2 layers). In conclusion, the different collagen composition of each layer was able to spatially direct BMSC differentiation in a localized manner within the scaffold. Ongoing work is evaluating the synergistic effect between growth factor functionalized within the fibrocartilage and tendon-like layers for improved BMSC differentiation. Overall, these scaffolds hold promising potential in developing novel and more efficient strategy towards enthesis regeneration

Malreduction of the syndesmosis is a poor prognosticator following ankle fracture and has been documented in as many as 52% of patients following fracture fixation. The current standard for assessment of reduction of the syndesmosis is bilateral computed tomography (CT) scan of the ankle. Multiple radiographic parameters are utilized to define malreduction, however, there has been limited investigation into the accuracy of these measurements to differentiate malreduction from inherent anatomical asymmetry. The purpose of this study was to identify the prevalence of positive malreduction standards within the syndesmosis of native, uninjured ankles. Bilateral lower extremity CT scans including ankles were screened. Studies were excluded if the patient was skeletally immature, had pathology below the knee or if they had congenital neuromuscular syndromes. The resulting cohort consisted of 207 patients. The indication for bilateral CT scan was femoral acetabular impingement in 110 patients (53%), rotation assessment following arthroplasty in 32 patients (15%), rotation assessment following femoral fracture in 30 patients (14%), rotational assessment for patellar instability in 30 patients (14%) and five miscellaneous indications (2%). Fifty patients were reviewed by three observers independently and to determine inter-observer reliability. A single observer repeated the measurements within the same cohort four weeks later to evaluate intra-observer reliability. Three observers then measured the anterior syndesmotic distance, posterior syndesmotic distance, central syndesmotic distance, fibular rotation and sagittal fibular translation at 1cm from the distal tibial articular surface. Overall side to side variability between the left and right ankle were assessed. Previously studied malreduction standards were evaluated. These included: anterior to posterior syndesmotic distance > 2mm, central syndesmotic difference > 1.5mm, average syndesmotic distance > 2mm, fibular rotational difference > 10o and sagittal translational difference > 2mm. The inter- and intra-observer reliability was good to excellent for anterior, posterior and central syndesmotic distance, and fibular rotation measurements. Sagittal fibular translation had an ICC of 0.583, and thus was only of fair reliability. Side to side comparison revealed statistically significant difference in only anterior syndesmotic difference (p=0.038). A difference of anterior to posterior syndesmotic distance of greater than 2mm was observed in 43 patients (20.2%). Thirty eight patients (17.8%) had a central syndesmotic difference of greater than 1.5mm. A fibular rotational difference of greater than 10o was observed in 49 patients (23%). The average difference between the anterior and posterior syndesmosis was greater than 2mm in 17 patients (8.2%). Nine patients (4.2%) had sagittal translation of greater than 2mm. Eighty one patients (39%) demonstrated at least one parameter beyond previously set standards for malreduction. Only one parameters was anomalous in 54 patients (26%), 18 patients (8%) had two positive parameters, while eight patients (4%) had three. One patient was asymmetrical in all measured parameters. In this study there was no statistically significant asymmetry between ankles. However, 39% of native syndesmoses would be classified as malreduced on CT scan using previously studied malreduction limits. Current radiographic parameters are not sufficient to differentiate mild inherent anatomical asymmetry from malreduction of the syndesmosis

Osteoblast progenitor cells can be isolated from human bone marrow and on an appropriate carrier following differentiation into osteoblasts a bone block could be formed. This supply of autologous, osteoinductive bone graft substitute would have significant implications for clinical use. The aim of the study was to assess whether osteoblast progenitor cells isolated from human bone marrow, seeded onto porous hydroxyapatite (HA) blocks adhere, proliferate and differentiate into osteoblasts under the influence of HA alone. After informed consent, bone marrow was aspirated from the iliac crest of 8 patients. The osteoblast progenitor cells were separated from the haematological cells and cultured in vitro. Evidence for the osteoblast progenitor nature of the cells was obtained by adding osteogenic supplements: dexamethasone, ascorbic acid and b-glycophosphate, and comparing alkaline phosphatase (ALP) and osteocalcin expression with that of unstimulated cells. Undifferentiated osteoblast progenitor cells were seeded at a density of 2x10 . 6. cells/porous HA cylindrical block (8 x 8 x10 mm). The cell adhesion to the HA was observed, and proliferation and ALP expression was measured over 15 days. In monolayer culture the isolated bone marrow cells were morphologically identified as mesenchymal stem cells. When osteogenic supplements were added the phenotype became consistent with the morphology of osteoblastic cells, and the ALP expression was significantly higher (P< 0.05) after 5 days in culture compared with cells that had not been stimulated to differentiate. On the HA osteoblast progenitor cells were adherent and became more osteoblastic, being separated from the HA surface by an osteoid matrix layer on electron microscopy. The ALP expression by these cells increased significantly (P< 0.05) over the 15 day culture period. Bone marrow contains mesenchymal stem cells with osteogenic potential that are known as osteoblast progenitor cells. In this study we have shown that osteoblast progenitor cells can be isolated from human bone marrow and will adhere to and proliferate on HA blocks in vitro, and differentiate into osteoblasts spontaneously under the influence of the HA scaffold. These constructs could be used as osteoinductive bone grafts

Introduction: We have investigated the accuracy of a serological marker to distinguish between septic and aseptic loosening of Total Hip Replacements (THR). We present the preliminary results of our on-going prospective study. Methods: After obtaining Ethical Committee approval, 46 patients were collected in 3 groups; “control” primary THR, revision THR for aseptic loosening, and revision THR for infection. Serum IgG responses to an exocellular bacterial antigen (Lipid S) were determined by enzyme-linked immunosorbent assay (ELISA). Results: Our results show that the test can accurately differentiate between the patients with infected joint replacements and the control group. The test, to date, has a specificity of 93% and a sensitivity of 100%. Discussion and Conclusion: This simple and cheap test can reliably assist in the accurate evaluation of a painful hip arthroplasty, and planning for revision surgery. It will also be useful in the management of patients in whom the microbiology results are either negative or based on a single isolate of an organism, which may be either a contaminant or a possible pathogen. This, in turn, would have implications on financial costs and the optimum use of available resources

Quantitative assessment of metastatic involvement of the bony spine is important for assessing disease progression and treatment response. Quantification of metastatic involvement is challenging as tumours may appear as osteolytic (bone resorbing), osteoblastic (bone forming) or mixed. This investigation aimed to develop an automated method to accurately segment osteoblastic lesions in a animal model of metastatically involved vertebrae, imaged with micro computed tomography (μCT).

Radiomics seeks to apply standardized features extracted from medical images for the purpose of decision-support as well as diagnosis and treatment planning. Here we investigate the application of radiomic-based features for the delineation of osteoblastic vertebral metastases. Osteoblastic lesions affect bone deposition and bone quality, resulting in a change in the texture of bony material physically seen through μCT imaging. We hypothesize that radiomics based features will be sensitive to changes in osteoblastic lesion bone texture and that these changes will be useful for automating segmentation.

Osteoblastic metastases were generated via intracardiac injection of human ZR-75-1 breast cancer cells into a preclinical athymic rat model (n=3). Four months post inoculation, ex-vivo μCT images (µCT100, Scanco) were acquired of each rodent spine focused on the metastatically involved third lumbar vertebra (L3) at 7µm/voxel and resampled to 34µm/voxel.

The trabecular bone within each vertebra was isolated using an atlas and level-set based segmentation approach previously developed by our group. Pyradiomics, an open source Radiomics library written in python, was used to calculate 3D image features at each voxel location within the vertebral bone. Thresholding of each radiomic feature map was used to isolate the osteoblastic lesions.

The utility of radiomic feature-based segmentation of osteoblastic bone tissue was evaluated on randomly selected 2D sagittal and axial slices of the μCT volume. Feature segmentations were compared to ground truth osteoblastic lesion segmentations by calculating the Dice Similarity Coefficient (DSC). Manually defined ground truth osteoblastic tumor segmentations on the μCT slices were informed by histological confirmation of the lesions.

The radiomic based features that best segmented osteoblastic tissue while optimizing computational time were derived from the Neighbouring Gray Tone Difference Matrix (NGTDM). Measures of coarseness yielded the best agreement with the manual segmentations (DSC=707%) followed by contrast, strength and complexity (DSC=6513%, 5428%, and 4826%, respectively).

This pilot study using a radiomic based approach demonstrates the utility of the NGTDM features for segmentation of vertebral osteoblastic lesions. This investigation looked at the utility of isolated features to segment osteoblastic lesions and found modest performance in isolation. In future work we will explore combining these features using machine learning based classifiers (i.e. decision forests, support vector machines, etc.) to improve segmentation performance.

Introduction and Aims: We have investigated the accuracy of a serological marker to distinguish between septic and aseptic loosening of Total Hip Replacements (THR). We present the preliminary results of our ongoing prospective study. Method: After obtaining Ethical Committee approval, 46 patients were collected in three groups: ‘control’ primary THR, revision THR for aseptic loosening and revision THR for infection. Serum IgG responses to an exocellular bacterial antigen (LipidS) were determined by enzyme-linked immunosorbent assay (ELISA). Results: Our results show that the test can accurately differentiate between the patients with infected joint replacements and the control group. The test, to date, has a specificity of 93% and a sensitivity of 100%. Conclusions: This simple and cheap test can reliably assist in the accurate evaluation of a painful hip arthroplasty, and planning for revision surgery. It will also be useful in the management of patients in whom the microbiology results are either negative or based on a single isolate of an organism, which may be either a contaminant or a possible pathogen. This, in turn, would have implications on financial costs and the optimum use of available resources

Introduction: We have investigated the accuracy of a serological marker to distinguish between septic and aseptic loosening of Total Hip Replacements (THR). We present the preliminary results of our on-going prospective study. Methods: After obtaining Ethical Committee approval, 46 patients were collected in 3 groups; “control” primary THR, revision THR for aseptic loosening, and revision THR for infection. Serum IgG responses to an exocellular bacterial antigen (Lipid S) were determined by enzyme-linked immunosorbent assay (ELISA). Results: Our results show that the test can accurately differentiate between the patients with infected joint replacements and the control group. The test, to date, has a specificity of 93% and a sensitivity of 100%. Clinical Relevance: This simple and cheap test can reliably assist in the accurate evaluation of a painful hip arthroplasty, and planning for revision surgery. It will also be useful in the management of patients in whom the microbiology results are either negative or based on a single isolate of an organism, which may be either a contaminant or a possible pathogen. This, inturn, would have implications on financial costs and the optimum use of available resources

Background

Establishing the diagnosis in a child presenting with an atraumatic limp can be difficult. Clinical prediction algorithms have been devised to distinguish septic arthritis (SA) from transient synovitis (TS). Within Europe measurement of the Erythrocyte Sedimentation Rate (ESR) has largely been replaced with assessment of C-Reactive Protein (CRP) as an acute phase protein. We produce a prediction algorithm to determine the significance of CRP in distinguishing between TS and SA.

We found that adipose stem cells are poorly differentiated into bone and that their ability to differentiate into bone varies from cell line to cell line. The osteogenic differentiation ability of the adipose stem cell lines was distinguished through Alzarin Red Staining, and the cell lines that performed well and those that did not were subjected to RNA-seq analysis. The selected gene GSTT1 (glutathione S-transferase theta-1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. The purpose of this study is to treat avascular necrosis and bone defect by improving bone regeneration with adipose stem cells introduced with a new GSTT1 gene related to osteogenic differentiation of adipose stem cells. In addition, the GSTT1 gene has the potential as a genetic marker that can select a specific cell line in the development of an adipose stem cell bone regeneration drug. Total RNA was extracted from each sample using the TRIzol reagent. Its concentration and purity were determined based on A260 and A260/A280, respectively, using a spectrophotometer. RNA sequencing library of each sample was prepared using a TruSeq RNA Library Prep Kit. RNA-seq experiments were performed for hADSCs. Cells were transfected with either GSTT1 at 100 nM or siControl (scramble control) by electroporation using a 1050 pulse voltage for 30 ms with 2 pulses using a 10 μl pipette tip. The purpose of this study is to discover genetic markers that can promote osteogenic differentiation of adipose stem cells (hADSCs) through mRNA-seq gene analysis. The selected GSTT1 gene was found to be associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic conditions. In this study, GSTT1 transgenic adipose stem cells could be used in regenerative medicine to improve bone differentiation. In addition, the GSTT1 gene has important significance as a marker for selecting adipose stem cells with potential for bone differentiation in the development of a therapeutic agent for bone regeneration cells

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2. Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining. Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation. Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S

Nitric oxide is a free radical which in vivo is solely produced during the conversion of the amino acid arginine into citrulline by nitric oxide synthase enzymes. Recently, the importance of nitric oxide on inflammation and bone metabolism has been investigated. However, the knowledge regarding possible in vitro effects of arginine supplementation on chondrogenic differentiation is limited. ATDC5, a cell line which is derived from mouse teratocarcinoma cells and which is characterized as chondrogenic cell line, were proliferated in Dulbecco's Modified Eagle Medium (DMEM)/F12 and subsequently differentiated in proliferation medium supplemented with insulin, transferrin and sodium-selenite and where arginine was added in four different concentrations (0, 7.5, 15 and 30 mM). Samples were harvested after 7 or 10 days and were stored at −80 °C for subsequent RNA isolation for qPCR analysis. To determine chondrogenic differentiation, Alcian Blue staining was performed to stain the proteoglycan aggrecan, which is secreted by differentiated ATDC5 cells. All measurements were performed in triplo. Alcian Blue staining showed a qualitative increase of proteoglycan aggrecan secretion in differentiated ATDC5 cells after treatment with 7 and 15 mM arginine, with additional increased expression of ColII, ColX, Bmp4 and Bmp6. Treatment with 30 mM arginine inhibited chondrogenic differentiation and expression of aforementioned genes, however, Cox-2 and Vegfa gene expression were increased in these samples. Bmp7 was not significantly expressed in any experimental condition. The obtained results are suggestive for a dose-dependent effect of arginine supplementation on chondrogenic differentiation and associated gene expression, with 7.5 and 15 mM as most optimal concentrations and implications for apoptosis after incubation with 30 mM arginine. A future recommendation would be to investigate the effects of citrulline in a similar experiment, as this shows even more promising results to enhance the nitric oxide metabolism in sepsis and bone healing

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel. ASCs at 2*10. 6. cells/mL were embedded in a 3D VitroGel RGD. ®. hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated. Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis. These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE. Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis)

Mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues in response to injury, such as fracture or other tissue injury. Bone marrow and adipose tissue are the major sources of MSCs. Previous studies suggested that the regenerative activity of stem cells can be enhanced by exposure to tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells-derived mesenchymal-like progenitors (hiPSCs-MPs) can enhance the regenerative potential of human bone marrow mesenchymal stromal cells (hBMSCs). ECM was engineered from hiPSC-MPs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. hBMSCs were cultured on the engineered ECM, and differentiated into osteogenic, chondrogenic and adipogenic lineages. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularization of ECM resulted in efficient cell elimination, as observed in our previous studies. Cultivation hBMSCs on the ECM in osteogenic medium significantly increased hBMSC growth, collagen deposition and alkaline phosphatase activity. Furthermore, expression of osteogenic genes and matrix mineralization were significantly higher compared to plastic controls. Chondrogenic micromass culture on the ECM significantly increased cell growth and expression of chondrogenic markers, including glycosaminoglycans and collagen type II. Adipogenic differentiation of hBMSCs on the ECM resulted in significantly increased hBMSC growth, but significantly reduced lipid vacuole deposition compared to plastic controls. Together, our studies suggest that BMSCs differentiation into osteogenic and chondrogenic lineages can be enhanced, whereas adipogenic activity is decreased by the culture on engineered ECM. Contribution of specific matrix components and underlying mechanisms need to be further elucidated. Our studies suggest that the three-lineage differentiation of aged BMSCs can be modulated by culture on hiPSC-engineered ECM. Further studies are aimed at scaling-up to three-dimensional ECM constructs for osteochondral tissue regeneration

Extensive bone defects, caused by severe trauma or resection of large bone tumors, are difficult to treat. Regenerative medicine, including stem cell transplantation, may provide a novel solution for these intractable problems and improve the quality of life in affected patients. Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. However, the osteogenic potential of ASCs is lower than that of bone marrow-derived stromal/stem cells. To address this disadvantage, our group has employed various methods to enhance osteogenic differentiation of ASCs, including factors such as bone morphogenetic protein or Vitamin D, coculture with bone marrow stem cells, VEGF transfection, and gene transfer of Runx-2 and osterix. Recently, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation while GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1- transfected ASCs. GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones

Cell-based therapies offer a promising strategy to treat tendon injuries and diseases. Both mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) are good candidates for such applications due to their self-renewing and differentiation capacity. However, the translation of cell-based therapies from bench to bedside can be hindered by the use of animal-derived components in ancillary materials and by the lack of standardised media and protocols for in vitro tenogenic differentiation. To address this, we have optimized animal component-free (ACF) workflows for differentiating human MSCs and PSCs to tenocyte-like cells (TLCs) respectively. MSCs isolated from bone marrow (n = 3) or adipose tissue (n = 3) were expanded using MesenCult™-ACF Plus Culture Kit for at least 2 passages, and differentiated to TLCs in 21 days using a step-wise approach. Briefly, confluent cultures were treated with an ACF tenogenic induction medium for 3 days, followed by treatment with an ACF maturation medium for 18 days. Monolayer cultures were maintained at high density without passaging for the entire duration of the protocol, and the medium was changed every 2 – 3 days. In a similar fashion, embryonic (n = 3) or induced PSCs (n = 3) were first differentiated to acquire a mesenchymal progenitor cell (MPC) phenotype in 21 days using STEMdiff™ Mesenchymal Progenitor Kit, followed by the aforementioned tenogenic protocol for an additional 21 days. In all cases, the optimized workflows using ACF formulations consistently activated a tenogenic transcriptional program, leading to the generation of elongated, spindle-shaped tenomodulin-positive (TNMD+) cells and deposition of an extracellular matrix predominantly composed of collagen type I. In summary, here we describe novel workflows that can robustly generate TLCs from MSCs and hPSC-derived MPCs for potential translational applications

Low back pain (LBP) is a worldwide leading cause of disability. Treatment of intervertebral disc (IVD) with stem cells has been used on degenerate discs (IDD), cause of around 40% of LBP cases. Despite pain reduction, clinical studies' follow-up have not shown a structural IVD improvement. A valid alternative may be the use of notocordal cells (NC) or their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC). MEPC derived from iPSC were developed during the iPSpine project (# 825925), thawed, plated for 24h on laminin and labeled with PKH26. Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 degenerated discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and one after 30 days. Clinical parameters were collected to evaluate the safety of treatment. Discs were analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated. At 7 days from treatment, both sheep lost about 20% of body weight. Only in discs treated with the highest dose PKH26 stained cells were alive up to 30 days. These cells turn out to be: proliferating (PCNA); positive for Brachyury, cytokeratin 8/18/19 and Foxa2; positive for SOX17 in a small percentage. This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive up to 30 days and differentiate within the disc predominantly towards the notocordal phenotype

Low back pain (LBP) is the leading cause of disability worldwide. Recently, treatment of the intervertebral disc (IVD) with stem cells has been used for the treatment of degenerate discs (IDD) which cause at least the 40% of LBP cases. Despite pain reduction, follow-up in clinical studies have not shown an improvement in the structural integrity of IVD. A valid alternative could be the use of progenitor disc cells (notocordal cells, NC) or of their precursors. Mesendoderm progenitor cells (MEPC) have the ability to replicate and differentiate toward NC. In this preliminary study we evaluated in a preclinical large animal IDD model the viability and NC differentiation of MEPC derived from induced pluripotent stem cells (iPSC). MEPC, derived from iPSC and developed during the iPSpine project (# 825925), were thawed and plated on laminin for 24h and labeled with PKH26. Two adult sheep were subjected to nucleotomy of five lumbar discs for the induction of IDD. After 5 weeks, 3 of the 5 degenerate discs were treated with MEPC at 3 different doses (low, medium and high). One sheep was sacrificed after 7 days and the other after 30 days from the treatment injection procedure. Clinical parameters were collected to evaluate the safety of treatment. Discs were paraffin embedded and analysed using histological techniques. Survival (PKH26), proliferation (PCNA), notocordal cell differentiation (Brachyury, Cytokeratin 8/18/19, Sox9, Foxa2) and endodermal differentiation (Sox17) were evaluated. After the injection of the cells, both sheep lost about 20% of body weight. The analysis showed that only in discs treated with the highest dose the PKH26 stained cells resulted alive after 30 days from the procedure. These cells turn out to be:. -. in proliferation (PCNA). -. positive for Brachyury, cytokeratin 8/18/19 and Foxa2. -. a small percentage positive for SOX17. This preliminary study shows that MEPC, derived from iPSC and injected into ovine discs degenerated by nucleotomy, are able to survive 30 days from treatment and differentiate within the disc predominantly towards the notocordal phenotype

Articular cartilage is a relatively hypoxic tissue with a unique extracellular matrix that is enriched with cations, resulting in an elevated interstitial fluid osmolarity. Several biomechanical and physicochemical stimuli are reported to influence chondrocyte metabolism. For regenerative in vitro applications, increasing the extracellular osmolarity above plasma level to more physiological valuesinduces chondrogenic marker expression and the differentiation of chondroprogenitor cells. Calcineurin inhibitor FK506 modulates the differentiation of primary chondrocytes under such conditions and its effect on cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling will be described. Supraphysiological osmolarity compromises chondrocyte proliferation, while physosmolarity or FK506 did not. Rather, the combination of the latter increased proteoglycan and collagen expression in chondrocytesin vitro and in situ, affecting expression of TGF-β-inducible protein TGFBI and chondrogenic (SOX9, Col2) as well as terminal differentiation markers (e.g., Col10). Surprisingly, expression of particularly minor collagens (e.g., Col9, Col11) was improved. Physiological osmolarity seems to promote terminal chondrogenic differentiation of progenitor cells through sensitization of TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 seems to enhance signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our data help explaining seemingly contradictory earlier findings and potentially benefit future cell-based cartilage repair strategies

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