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

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

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

Bone defects and fractures, caused by injury, trauma or tumour resection require hospital treatment and temporary loss of mobility, representing an important burden for societies and health systems worldwide. Autografts are the gold standard for promoting new bone formation, but these may provide insufficient material and lead to donor site morbidity and pain. We previously showed that Fibrinogen (Fg) scaffolds promote bone regeneration in vivo (1), and that modifying them with 10mM of Magnesium (Mg) ions modulates macrophage response in vitro and in vivo (2). Also, we showed that Extracellular Vesicles (EV) secreted by Dendritic Cells (DC) recruit Mesenchymal Stem/Stromal Cells (MSC)(3). Herein, we aim to functionalize FgMg scaffolds with DC-EV, to promote recruitment and osteogenic differentiation of MSC. Scaffolds were produced by freeze-drying (2). Ethical permission was sought for all studies. Primary human peripheral blood monocyte-derived DC were cultured, their secreted EV were isolated by differential (ultra)-centrifugation and characterised by transmission electron microscopy and nanoparticle tracking analysis (3). Bone marrow MSC were used to determine the impact of EV-functionalized scaffolds through migration assays and their osteogenic differentiation was assessed by Alizarin Red staining. Fg and FgMg scaffolds functionalized with EV were characterized. Fg and FgMg scaffolds functionalized with DC-secreted EV were more efficient at recruiting MSC than scaffolds alone. MSC cultured on FgMg scaffolds showed significantly increased calcium deposits, in comparison with those cultured on Fg scaffolds. Fg scaffold modification by Mg promotes MSC osteogenic differentiation, while their functionalization with DC-secreted EV acts to promote MSC recruitment. This renders the FgMg-EV functionalized scaffolds an attractive material to promote new bone formation. Acknowledgments: Work funded by Orthoregeneration Network (ON Pilot Grant Spine 2021, EVS4Fusion). MCT supported by ERASMUS+ program

The growing number of non-union fractures in an aging population has increased the clinical demand for tissue-engineered bone. Electrical stimulation (ES) has been described as a promising strategy for bone regeneration treatments in several clinical studies. However the underlying mechanism by which ES augments bone formation is still poorly understood and its use in bone tissue engineering (BTE) strategies is currently underexplored. Additive manufacturing (AM) technologies (Fused Deposition Modeling/3D Printing) have been widely used in BTE due to their ability to fabricate scaffolds with a high control over their structural and mechanical properties in a reproducible and scalable manner. Thus, in this work, we combined AM methods with conductive biomaterials and ES to enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) envisaging improved BTE strategies. First, we started by developing AM-based electro-bioreactor devices containing medical-grade electrodes (stainless steel and Ti6Al4V) to apply ES to monolayer 2D cultures and 3D cell-seeded scaffolds. Computer modeling(Finite Element Analysis-FEA) was employed to predict the magnitude/distribution of electrical fields within the ES devices and along the different conductive scaffolds. Prior to scaffold culture, 5 different ES protocols were tested in terms of their ability to promote hBMSCs proliferation and osteogenic differentiation in 2D cultures. The best performance ES protocol was then used in two different AM-based BTE strategies: 1) Two different conductive scaffolds (conductive poly lactic acid (PLA) and titanium) were seeded with hBMSCs and cultured for 21 days under osteogenic medium conditions with and without ES and their biological performance was evaluated in comparison to non-conductive standard PLA scaffolds; 2) Different PEDOT:PSS-based coating solutions were screened to obtain PEDOT:PSS/Gelatin-coated 3D polycaprolactone (PCL) scaffolds with a high(11 S.cm. -1. ) and stable electroconductivity. When cultured under ES, PEDOT:PSS/Gelatin-PCL scaffolds enhanced significantly hBMSCs osteogenic differentiation and mineralization(calcium deposition), highlighting their potential for BTE applications. Acknowledgements: Funding received from FCT through projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), and to the institutions iBB (UIDB/04565/2020), CDRSP (UIDB/04044/2020) and Associate Laboratory i4HB (LA/P/0140/2020)

Critical size bone defects pose a serious clinical problem, as the intrinsic healing capacity of bone fails due to the size of the defect. Bone healing might be aided by addition of 1,25(OH)2 vitamin D3 (vitD3) to bone tissue engineering scaffolds. VitD3can promote osteogenic differentiation of human stem cells such as adipose stem cells (hASCs), which is a clinical-relevant source of mesenchymal stem cells. However, it is unknown which release kinetics of vitD3, i.e. short or sustained release from scaffolds, leads to the most optimal osteogenic differentiation of hASCs. We hypothesized that sustained release of vitD3 leads to more osteogenic differentiation of hASCs than shorter applications. hASCs (1×105, passage 3–4) were seeded on 20 ± 1 mg of calcium phosphate particles (day 0), cultured for 20 days, and treated with a total amount of 124 ng vitD3. This treatment was provided either during 30 min before seeding (pre-incubation, short stimulation: [200 nM]), after seeding, over the first 2 days (burst- release high: [100 nM]), or over the total culture period of 20 days (sustained-release: [10 nM]). In the extra condition: burst-release low the hASCs were treated for 2 days after seeding with 6.2 ng vitD3 ([10 nM]) per day. Live/dead staining followed by fluorescent microscopy showed that hASCs attached to the calcium phosphate particles and were mostly viable (±75 %) at day 2. VitD3 applied for any duration did not affect the proliferation of hASCs at day 7 and day 20, measured with an alamar blue assay. At day 7, sustained-release increased the release of active alkaline phosphatase on average by 3.5-fold, compared to all the other conditions. At day 20, this was increased 4.3-fold. At both day 7 and day 20 total protein levels were similar in all conditions. Our results suggest that sustained release of VitD3 from bone tissue engineered scaffolds may be beneficial for the osteogenic differentiation of human stem cells for the treatment of critical bone size defects

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity. Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining. HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts. Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity

Bone is a dynamic tissue that undergoes continuous mechanical forces. Mechanical stimuli applied on scaffolds resembling a part of the human bone tissue affects the osteogenesis [1]. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a piezoelectric material that responds to mechanical stimulation producing an electrical signal, which in turn promotes the osteogenic differentiation of bone-forming cells by opening voltage-gated calcium channels [2]. In this study we examined the biological behavior of pre-osteoblastic cells seeded onto lyophilized piezoelectric PEDOT-containing scaffolds applying uniaxial compression. Two different concentrations of PEDOT (0.10 and 0.15% w/v) were combined with a 5% w/v poly(vinyl alcohol) (PVA) and 5% w/v gelatin, casted into wells, freeze dried and crosslinked with 2% v/v (3-glycidyloxypropyl)trimethoxysilane and 0.025% w/v glutaraldehyde. The scaffolds were physicochemically characterized by FTIR, measurement of the elastic modulus, swelling ratio and degradation rate. The cell-loaded scaffolds were subjected to uniaxial compression with a frequency of 1 Hz and a strain of 10% for 1 h every second day for 21 days. The loading parameters were selected to resemble the in vivo loading situation [3]. Cell viability and morphology on the PEDOT/PVA/gelatin scaffolds was determined. The alkaline phosphatase (ALP) activity, the collagen and calcium production were determined. The elastic modulus of PEDOT/PVA/gelatin scaffolds ranged between 1 and 5 MPa. The degradation rate indicates a mass loss of 15% after 21 days. The cell viability assessment displays excellent biocompatibility, while SEM images display well-spread cells. The ALP activity at days 3, 7 and 18 as well as the calcium production are higher in the dynamic culture compared to the static one. Moreover, energy dispersive spectroscopy analysis revealed the presence of calcium phosphate in the extracellular matrix after 14 days. The results demonstrate that PEDOT/PVA/gelatin scaffolds promote the adhesion, proliferation, and osteogenic differentiation of pre-osteoblastic cells under mechanical stimulation, thus favoring bone regeneration

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

The goal was to analyze the cellular response, specifically the osteogenic capacity, of titanium (Ti) implants harbouring a novel laserbased-surface-structure with the overall aim: augmented osteointegration. Surface micro-/nanoproperties greatly influence cell behaviour at the tissue-implant-interface and subsequent osteointegration. We investigated Ti-materials subjected to a specially developed shifted-Laser-Surface-Texturing (sLST) technology and compared them to a standard roughening-technique (sand-blasting-acid-etching, SLA). The biological response was evaluated with hMSCs, which are naturally available at the bone-implant-interface. We hypothesized: the novel surface is beneficial for our three different (young/healthy-YH; aged/healthy-AH;aged/osteoporotic-OP) cohorts. The sLST was performed using a SPI-G3-series laser (beam-wavelength=1064nm, pulse-duration=200ns). For the SLA surface, Ti was sandblasted, afterwards acid-etched (HCl/H2SO4). Three different hMSC cohorts were studied: YH: n=6,29±6; AH: n=5,79±5; OP: n=5,76±5 years (osteoporosis confirmed via DEXA-scan). OP hMSCs show e.g. ColI-deficient-matrix and decreased mineralization. Cells were examined for survival, cell proliferation and cytoskeleton arrangement. Osteogenic differentiation was carried out over 21 days, matrix mineralization was validated with Alizarin-Red-S-staining and quantification. Laser-texturing generated precisely the desired microgeometry. On nanostructural level, differently-sized Ti-droplets were formed stochastically by laser-induced-Ti-plasma. Live/dead-/Actin-stainings showed comparable results for all cohorts and surfaces in terms of survival and cell shape. On Ti-materials, cell growth showed no significant difference between the 3 cohorts. Alizarin quantification revealed the highest levels on laser-textured-surfaces; highest value for YH, followed by AH, lastly OP; no significance between AH/YH, but between OP/YH (p<0.0001). However, mineralization of all cohorts cultured on laser-textured-surfaces increased significantly (p<0.0001) compared to respective SLA-group, with >20fold higher value in the OP-cohort (AH:11fold, YH:6fold). The data proves the biocompatibility of the laser-structured-Ti for young+aged cohorts. Osteogenic differentiation was significantly augmented on laser-treated-Ti. Most intriguingly, OP-donors could reach manifold increased mineralization, suggesting the novel laser texturing can counteract the osteoporotic phenotype. As osteogenesis-enhancing capacities may be related to mechanisms controlling cellular shape/fate, further investigations referring to this are currently ongoing. In conclusion, our laser-textured-Ti-materials are safe, can have a demand-oriented designer-surface-topography and represent a great potential for development into next-generation-implants suitable for different patient-cohorts, especially osteoporosis patients

Minimally invasive surgery for the restoration of bone tissues lost due to diseases and trauma is preferred by the health care system as the related costs are continuously increasing. Recently, efforts have been paid to optimize injectable calcium phosphate (CaP) cements which have been recognized as excellent alloplastic material for osseous augmentation because of their unique combination of osteoconductivity, biocompatibility and mouldability. The sol-gel synthesis approach appears to be the most suitable route towards performing injectable calcium phosphates. Different strategies used to prepare bioactive and osteoinductive injectable CaP are reported. CaP gels complexed with phosphoserine-tethered poly(ε-lysine) dendrons (G3-K PS) designed to interact with the ceramic phase and able to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) is discussed. Recently, attention has been given to the modification of hydroxyapatite with Strontium (Sr) due to its dual mode of action, simultaneously increasing bone formation (stimulating osteoblast differentiation) while decreasing bone resorption (inhibiting osteoclast differentiation). The effect of systems based on strontium modified hydroxyapatite (Sr-HA) at different composition on proliferation and osteogenic differentiation of hMSC is described. One more approach is based on the use of antimicrobial injectable materials. It has been demonstrated that some imidazolium, pyridinium and quaternary ammonium ionic liquids (IL) have antimicrobial activity against some different clinically significant bacterial and fungal pathogens. Here, we report several systems based on IL at different alkyl-chain length incorporated in Hydroxyapatite (HA) through the sol-gel process to obtain an injectable material with simultaneous opposite responses toward osteoblasts and microbial proliferation

Millions of patients each year suffer from challenging non-healing bone defects secondary to trauma or disease (e.g. cancer, osteoporosis or osteomyelitis). Tissue engineering approach to non-healing bone defects has been investigated over the past few decades in a search for a novel solution for critical size bone defects. The success of the tissue engineering approach relies on three main pillars, the right type of cells; and appropriate scaffold; and a biologically relevant biochemical/ biophysical stimuli. When it comes to cells the mesodermal origin of mesenchymal stem cells and its well demonstrated multipotentiality makes it an ideal option to be used in musculoskeletal regeneration. For the presented set of experimental assays, fully characterised (passage 3 to 5)ovine adipose-derived mesenchymal stems cells (Ad-MSC) were cultured either in growth medium (GM) consisting of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and 1% penicillin-streptomycin as a control or in osteogenic differentiation medium (DM), consisting of GM further supplemented with L- ascorbic acid (50 μg/ml), β-glycerophosphate (10 mM) and dexamethasone (100nM). Osteogenic differentiation was assessed biochemically by quantifying alkaline phosphatase (ALP) enzyme activity and alizarin red staining after 3, 7, 14 and 21 days in culture (where 1×105 cells/well were seeded in 24 well-plate, n=6/media type/ time point). Temporal patterns in osteogenic gene expression were quantified using real-time PCR for Runx-2, osteocalcin (OC), osteonectin (ON) and type 1 collagen (Col 1) at days 7, 15 and 21 (where 1×105 cells were seeded in T25 cell culture flasks for RNA extraction, n= 4 / gene/ media type/time point). The morphology of osteogenic cells was additionally evaluated by scanning electron microscopy (SEM) of cells seeded at low-density (1×102 cells) on glass coverslips for 2 weeks in GM or DM. The level of ALP activity of cells grown in osteogenic DM was significantly higher than the control growing in the standard growth medium (p ≤ 0.05) at days 3, 7 and 14. At 21 days there was a sharp drop in ALP values in the differentiating cells. Mineralisation, as evidenced by alizarin red staining, increased significantly by day 14 and then peaked at day 21. Quantitative real-time PCR confirmed early increases in Runx-2, Col 1 and osteonectin, peaking in the second week of culture, while osteocalcin peaked at 21 days of culture. Taken as a whole, these data indicate that ovine-MSCs exhibit a tightly defined pathway of initial proliferation and matrix maturation (up to 14 days), followed by terminal differentiation and mineralisation (days 14 to 21). SEM analysis confirmed the flattened, roughened appearance of these cells and abandoned extracellular matrix which resembled mature osteoblasts. Given the ready availability of adipose tissues, the use of Ad-MSCs as progenitors for bone tissue engineering applications is both feasible and reasonable. The data from this study indicate that Ad-MSCs follow a predictable pathway of differentiation that can be tracked using validated molecular and biochemical assays. Additional work is needed to confirm that these cells are osteogenic in vivo, and to identifying the best combination of scaffold materials and cell culture techniques (e.g. static versus dynamic) to accelerate or stimulate osteogenic differentiation for bone tissue engineering applications

Objectives. Venous thromboembolism (VTE) is a major potential complication following orthopaedic surgery. Subcutaneously administered enoxaparin has been used as the benchmark to reduce the incidence of VTE. However, concerns have been raised regarding the long-term administration of enoxaparin and its possible negative effects on bone healing and bone density with an increase of the risk of osteoporotic fractures. New oral anticoagulants such as rivaroxaban have recently been introduced, however, there is a lack of information regarding how these drugs affect bone metabolism and post-operative bone healing. Methods. We measured the migration and proliferation capacity of mesenchymal stem cells (MSCs) under enoxaparin or rivaroxaban treatment for three consecutive weeks, and evaluated effects on MSC mRNA expression of markers for stress and osteogenic differentiation. Results. We demonstrate that enoxaparin, but not rivaroxaban, increases the migration potential of MSCs and increases their cell count in line with elevated mRNA expression of C-X-C chemokine receptor type 4 (CXCR4), tumor necrosis factor alpha (TNFα), and alpha-B-crystallin (CryaB). However, a decrease in early osteogenic markers (insulin-like growth factors 1 and 2 (IGF1, IGF2), bone morphogenetic protein2 (BMP2)) indicated inhibitory effects on MSC differentiation into osteoblasts caused by enoxaparin, but not by rivaroxaban. Conclusions. Our findings may explain the adverse effects of enoxaparin treatment on bone healing. Rivaroxaban has no significant impact on MSC metabolism or capacity for osteogenic differentiation in vitro. Cite this article: Dr H. Pilge. Enoxaparin and rivaroxaban have different effects on human mesenchymal stromal cells in the early stages of bone healing. Bone Joint Res 2016;5:95–100. DOI: 10.1302/2046-3758.53.2000595

Summary Statement. Low-intensity pulsed ultrasound (LIPUS) enhanced osteogenic differentiation of osteoprogenitor cells derived from mouse induced pluripotent cells (iPSCs) without embryoid body formation. Our findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs. Introduction. iPSCs represent a promising cell source for regenerative medicine such as bone regeneration because of their unlimited self-renewal property and ability of differentiation into all somatic cell types. Recently, we developed an efficient protocol for generating a highly homogeneous population of osteoprogenitor cells from embryonic stem cells by using a direct-plating method without EB formation step. It is well-recognised that LIPUS accelerates the fracture healing. There have been several reports showing that LIPUS stimulates the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. To date, effect of LIPUS on iPSCs remains unknown. In this study, we investigated in vitro effect of LIPUS on osteogenic differentiation of osteoprogenitor cells derived from mouse iPS cells via a direct-plating method. Methods. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates without feeders in MSC medium and FGF-2. Adherent fibroblastic cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. For osteogenic differentiation, DPCs were divided into two groups: (1) control group: DPCs cultured in osteogenic medium (OM) without LIPUS, and (2) LIPUS group: DPCs cultured in OM with LIPUS treatment. LIPUS was given through the bottom of the culture plates for 20 minutes daily. After 14-day culture, osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin red S staining. Expression of osteoblast-related genes, Rnux2 and ALP was also analyzed by real-time PCR. Results. Flow cytometry analysis revealed DPCs had similar characteristics to MSCs. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs, suggesting DPCs lost pluripotency. After 14-day osteogenic induction, ALP activity was shown to be higher in LIPUS group than control group on days 3 and 7. Real-time PCR analysis revealed that in LIPUS group, expression level of Runx2 on day 1 and that of ALP on days 3 and 5 were significantly up-regulated compared to control group. The quantity of calcium deposition measured by Alizarin red staining on day 14 was shown to be higher in LIPUS group than control group. Conclusion. The novel direct-plating method described here provides a significant technical advance over conventional methods of isolating iPSC-induced osteoprogenitor cells by avoiding the embryonic body formation that often leads to heterogeneous, variable, and unpredictable osteogenic differentiation. Our results demonstrated that osteogenic differentiation of osteoprogenitor cells from iPSCs was robustly increased by LIPUS treatment. LIPUS may be a promising enhancer of osteogenesis of iPSCs. These findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs

Introduction. Despite the high regenerative capacity of bone, large bone defects often require treatment involving bone grafts. Conventional autografting and allografting treatments have disadvantages, such as donor site morbidity, immunogenicity and lack of donor material. Bone tissue engineering offers the potential to achieve major advances in the development of alternative bone grafts by exploiting the bone-forming capacity of osteoblastic cells. However, viable cell culture models are essential to investigate osteoblast behavior. Three-dimensional (3D) cell culture systems have become increasingly popular because biological relevance of 3D cultures may exceed that of cell monolayers (2D) grown in standard tissue culture. However, only few direct comparisons between 2D and 3D models have been published. Therefore, we performed a pilot study comparing 2D and 3D culture models of primary human osteoblasts with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation. Patients and Methods. Primary human osteoblasts were extracted from femoral neck spongy bone obtained during surgery procedures. Primary human osteoblasts of three donor patients were cultured in monolayers and in three different 3D culture models: 1) scaffold-free cultures, also referred to as histoids, which form autonomously after multilayer release of an osteoblast culture; 2) short-term (10-day) collagen scaffolds seeded with primary human osteoblasts (HOB); 3) long-term (29-day) collagen scaffolds seeded with HOB. Expression levels of transcription factors RUNX2 and osterix, both involved in osteoblast differentiation, were investigated using quantitative PCR and immunohistochemical staining. Furthermore, markers of osteogenic differentiation were evaluated, such as alkaline phosphatase activity, osteocalcin expression, and mineral deposition, as well as the expression of collagen type I and fibronectin extracellular matrix proteins. Results. Cells of the same origin, which were cultivated in different culture models, showed varying expression levels with regard to transcription factors RUNX2 and osterix as well as osteogenic markers. Increased levels of transcription factor RUNX2 and the extracellular matrix protein fibronectin were observed in all 3D cell culture models compared to monolayers. Furthermore, long-term cultivated histoids showed increased levels of osteogenic late-stage marker osteocalcin and transcription factor osterix. Additionally, long-term collagen scaffolds seeded with HOB showed elevated levels of osteocalcin compared to monolayers and short-term scaffolds. Moreover, alkaline phosphatase activity and mineralization capacity were increased in histoids. Conclusion. Considering the complex biochemical interactions of cells with surrounding cells and the extracellular matrix in vivo, important biological properties are disregarded when cells are only studied in 2D study models. Hence, we compared different 3D HOB cell culture models to 2D HOB monolayers with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation in vitro. Our pilot study indicated that three-dimensional study models may promote osteogenic differentiation in vitro. Additionally, a beneficial effect of longer culture duration on osteogenic differentiation was observed. Hence, our findings emphasise the importance of dimension and culture duration when studying osteoblast function. Subsequent studies with higher sample sized may lead to the development of viable primary human osteoblast cell culture models for bone tissue engineering. Summary. Three-dimensional cell culture models of primary human osteoblasts (HOB), including collagen scaffolds and scaffold-free cultures, were compared to HOB monolayers with regard to osteogenic differentiation. Our study indicated that three-dimensional study models may promote osteogenic differentiation of HOB in vitro

Pulsed electromagnetic fields (PEMFs) have been considered a potential treatment modality for fracture healing. As bone fracture healing and osseointegration share the same biological events, the application of PEMF stimulation to facilitate the osseointegration process of orthopedic implants has been suggested. However, the mechanism of their action remains unclear. Mammalian target of rapamycin (mTOR) signaling may affect osteoblast proliferation and differentiation. This study aimed to assess the osteogenic differentiation of mesenchymal stem cells (MSCs) under PEMF stimulation and the potential involvement of mTOR signaling pathway in this process. PEMFs were generated by a novel miniaturized electromagnetic device (MED). Potential changes in the expression of mTOR pathway components, including receptors, ligands and nuclear target genes, and their correlation with osteogenic markers and transcription factors were analyzed. PEMF exposure increased cell proliferation, adhesion and osteogenic commitment of MSCs. Osteogenic-related genes were over-expressed following PEMF treatment. Our results confirm that PEMFs contribute to activation of the mTOR pathway via upregulation of the proteins AKT, MAPP kinase, and RRAGA, suggesting that activation of the mTOR pathway is required for PEMF-stimulated osteogenic differentiation. In summary, the findings of the present study revealed that MED-generated PEMFs stimulate osteogenic differentiation and the maturation of the adipose tissue-derived MSCs via activation of the mTOR pathways. Even though further research is required to determine an optimal stimulation timing and flux density both in-vitro and in-vivo, this study results may serve a source for an adjuvant therapy to improve orthopedic implant stability, longevity and enhance fracture healing

Intermittent parathyroid hormone (iPTH 1–34) increases bone formation via modelling and remodelling mechanisms and as such is used to treat osteoporosis. The actions of iPTH on mesenchymal stem cell (MSCs) may underpin a further treatment option. We isolated bone marrow derived MSCs from young (WT) and ovarectomized senile (OVX) rats, investigating the effect of intermittent and continuous PTH administration on migration to SDF-1, proliferation and osteogenic differentiation. MSCs were harvested from the femora of 6–10week old WT rats and 10–13month old OVX rats. Cells were cultured with 25,50 and 100nmMol of PTH 1–34 added to osteogenic media either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Alamar Blue- proliferation. Cells were seeded in a Boyden chamber to quantify SDF-1 migration. A student t-test was used to analyse results, and a p value<0.05 considered significant. ALP and Alizarin Red were significantly increased for WT and OVX groups at 50nmMol of iPTH. Continuous administration at all concentrations reduced calcium phosphate deposition by day 21 in all groups. In comparison to cells cultured in osteogenic media, 50nmMol of iPTH led to significantly higher ALP and Alizarin Red measurements up to days 10 and 7 respectively (figure 1). There was no change in proliferation between the groups, and PTH had no effect (figure 2.). WT MSCs not only had improved osteogenic differentiation, but also showed increased migration to SDF-1 in comparison to OVX groups. iPTH led to further increases in migration of both OVX and WT cells. iPTH increases the osteogenic differentiation and migration of MSCs from both young and ovarectomised rats, though this effect is not dose dependent. Ultimately, the role of iPTH on MSCs may lead to improved bone formation and cell homing capacity-particularly in the context of osteoporosis

Several synthetic polymers have been widely investigated for their use in bone tissue engineering applications, but the ideal material is yet to be engineered. Triazine-trione (TATO) based materials and their derivatives are novel in the field of biomedical engineering but have started to draw interest. Different designs of the TATO monomers and introduction of different chemical linkages and end-groups widens the scope of the materials due to a range of mechanical properties. The aim of our work is to investigate novel TATO based materials, with or without hydroxyapatite filler, for their potential in bone tissue engineering constructs. Initially the biocompatibility of the materials was tested, indirectly and directly, according to ISO standards. Following this the osteoconductive properties were investigated with primary osteoblasts and an osteoblastic cell line. Bone marrow derived mesenchymal stem cells were used to evaluate the osteogenic differentiation and consequently the materials potential in bone tissue engineering applications