Femoral Heads from Total Hip Arthroplasty as a Source of Adult Hematopoietic Cells.

Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3+, CD14+, and CD34+ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 107 (range, 1.62 × 105-2.52 × 108), and the median yield of CD34+ cells was 1.40 × 106 (range, 3.60 × 105-9.90 × 106). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.

Glucocorticoids induce femoral head necrosis in rats through the ROS/JNK/c-Jun pathway.

Osteonecrosis of the femoral head (ONFH) is a common clinical disease with a high disability rate. Apoptosis of osteoblasts caused by high-dose short-term or low-dose long-term glucocorticoid (GC) administration is the biological basis of steroid-induced avascular necrosis of the femoral head (SANFH). The pathogenesis of SANFH has not yet been fully elucidated, and there is currently a lack of effective clinical treatments. Here, we investigated the role of the reactive oxygen species (ROS)/JNK/c-Jun signaling pathway in SANFH. Dexamethasone (Dex) was used to induce apoptosis in osteoblasts, and this resulted in a significant increase in levels of p-JNK, p-c-Jun, Bax, caspase-3, caspase-9, cytochrome C, Beclin-1, and LC3, and a decrease in levels of P62 and Bcl-2. In addition, intracellular ROS levels were increased and mitochondrial membrane potential was decreased. Administration of 3-MA, an autophagy inhibitor, attenuated Dex-mediated changes in autophagy and apoptosis. A rat model of ONFH exhibited severe bone trabecular hollow bone pits along with a significant increase in femoral head cell apoptosis compared with the control group. Additionally, micro-CT analysis showed that both bone tissue content and femoral head integrity were significantly reduced in the ONFH group. Furthermore, 3-MA treatment decreased the effect of Dex on GC-induced ONFH and osteoblast apoptosis in rats and could counteract microstructure destruction due to femoral head necrosis. In summary, our data suggest that GC can induce osteoblast apoptosis and autophagy through the ROS/JNK/c-Jun signaling pathway, which contributes to ONFH.

ß-ecdysone promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: ß-ecdysone (ßEcd) has numerous pharmacological effects, although its role in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) has not yet been explored. METHODS: In cell experiments, BMSCs were induced to differentiate by osteogenic induction medium (OIM) or ßEcd. In animal experiments, an osteonecrosis of the femoral head (ONFH) rat model was established using lipopolysaccharide plus methylprednisolone and treating the rats with ßEcd. The osteogenic differentiation capacity of human BMSCs (hBMSCs) was analyzed by alkaline phosphatase and alizarin red S staining. Histopathological changes in rat femoral head tissues were observed by hematoxylin and eosin staining. The expression levels of RUNX2, COL1A1, OCN and phosphorylated Akt in BMSCs from rat femoral head tissues were measured by a quantitative real-time polymerase chain reaction or western blot analysis. RESULTS: Alkaline phosphatase activity and calcium nodules in the ßEcd-treated BMSC group dose-dependently increased compared to those in the control and OIM groups. The hematoxylin and eosin staining results indicated that femoral head tissues of ONFH rats showed typical osteonecrosis, which could be ameliorated by ßEcd. Western blot, quantitative real-time polymerase chain reaction and immunohistochemistry assays demonstrated that the expression levels of RUNX2, COL1A1 and OCN in hBMSCs and femoral head tissue models were obviously increased after ßEcd treatment, and phosphoinositide 3-kinase and Akt phosphorylation were also increased. CONCLUSIONS: ßEcd may be beneficial for the recovery of ONFH patients by accelerating osteogenic differentiation of BMSCs, which may be a novel therapy for related diseases.

The Proosteogenic and Proangiogenic Effects of Small Extracellular Vesicles Derived from Bone Marrow Mesenchymal Stem Cells Are Attenuated in Steroid-Induced Osteonecrosis of the Femoral Head.

Small extracellular vesicles (sEVs) derived from bone marrow mesenchymal stem cells (BMMSCs) from individuals with steroid-induced osteonecrosis of the femoral head (ONFH) have not been studied. The objective of the present study was to compare the proosteogenic and proangiogenic effects of sEVs derived from BMMSCs from rats with steroid-induced ONFH (oBMMSCs-sEVs) and sEVs derived from BMMSCs from normal rats (nBMMSCs-sEVs). BMMSCs were isolated from steroid-induced ONFH rats and healthy rats. sEVs were isolated and characterized by Western blotting analysis of exosomal surface biomarkers and by transmission electron microscopy. The impacts of nBMMSCs-sEVs and oBMMSCs-sEVs on the proliferation and osteogenic differentiation of BMMSCs were determined via cell proliferation assay, alizarin red staining, and alkaline phosphatase activity assay. Enzyme-linked immunosorbent assay and tube formation assay were conducted to investigate the effect of nBMMSCs-sEVs and oBMMSCs-sEVs on the angiogenic potential of human umbilical vein endothelial cells (HUVECs). The expression of relevant genes was detected by quantitative real-time polymerase chain reaction analysis, and the expression of ß-catenin was detected by immunofluorescence. Both nBMMSCs-sEVs and oBMMSCs-sEVs promoted proliferation, osteogenic differentiation, and ß-catenin expression of BMMSCs and enhanced angiogenesis of HUVECs. However, compared with nBMMSCs-sEVs, oBMMSCs-sEVs exhibited attenuated effects. Our findings indicated that the proosteogenic and proangiogenic effects of sEVs were partially attenuated in steroid-induced ONFH. Therefore, this study might offer guidance for the selection of source cells for sEV therapy in the future.

Decellularized human bone as a 3D model to study skeletal progenitor cells in a natural environment.

There has been an increasing interest in exploring naturally derived extracellular matrices as an material mimicking the complexity of the cell microenvironment in vivo. Bone tissue-derived decellularized constructs are able to preserve native structural, biochemical, and biomechanical cues of the tissue, therefore providing a suitable environment to study skeletal progenitor cells. Particularly for bone decellularization, different methods have been reported in the literature. However, the used methods critically affect the final ultrastructure and surface chemistry as well as the decellularization efficiency, consequently causing complications to draw conclusions and compare results in between studies. In this chapter, an optimized protocol for the preparation of human bone derived scaffolds is described, including processing techniques and further characterization methods, which allow the final construct to be recognized as a major platform for bone therapeutic and/or diagnostic applications.

Changed cellular functions and aberrantly expressed miRNAs and circRNAs in bone marrow stem cells in osteonecrosis of the femoral head.

The present study aimed to detect the correlations between altered cellular functions in bone marrow stem cells (BMSCs) and osteonecrosis of the femoral head (ONFH). By profiling the aberrant expression of miRNAs and circRNAs in BMSCs isolated from ONFH patients, the present study aimed to further explore the potential regulatory mechanisms of action of circRNAs in ONFH using integrated bioinformatics analysis. BMSCs were isolated from seven ONFH patients and seven controls. Cellular functions, including proliferation, apoptosis and differentiation, were compared. miRNA and circRNA sequencing were conducted using RNA samples of three ONFH patients and three controls to identify differentially expressed circRNAs and miRNAs. The expression of hsa_circ_0000219, hsa_circ_0004588 and hsa_circ_0005936 were validated by qPCR. Target miRNAs were also predicted and validated by qPCR and circRNA­miRNA co­expression networks were constructed. BMSCs of ONFH patients displayed decreased proliferation and increased apoptosis during in vitro culturing. In addition, reduced osteogenesis and enhanced adipogenesis were found in the ONFH group. A total of 129 miRNAs and 231 circRNAs were detected to be differentially expressed. The expression levels of hsa_circ_0000219, hsa_circ_0004588 and hsa_circ_0005936 were significantly decreased in BMSCs of ONFH patients. A number of target miRNAs related to cell proliferation, apoptosis and differentiation were predicted for hsa_circ_0000219 and hsa_circ_0005936. The expression levels of miR­144­3p and miR­1270 were found to be elevated in ONFH patients, which was consistent with miRNA sequencing data and competitive endogenous RNA hypothesis. Time­dependent expression patterns of hsa_circ_0000219, hsa_circ_0004588, hsa_circ_0005936, miR­144­3p and miR­1270 were also validated during osteogenic and adipogenic differentiation in BMSCs. The results of the present study substantiated the involvement of BMSCs in ONFH development. hsa_circ_0000219 and hsa_circ_0005936 may regulate the progression of ONFH by mediating the proliferation and differentiation of BMSCs by sponging miRNAs.

An Ex Vivo Bone Defect Model to Evaluate Bone Substitutes and Associated Bone Regeneration Processes.

The increased incidence of bone defects, especially in cases of comminuted fractures or bone tumor resections demands suitable bone grafts and substitutes. The aim of this study was to establish an ex vivo bone defect model to evaluate new bone substitutes and associated repair processes under controlled conditions. Femoral heads derived from patients undergoing total hip replacement were cut into cylinders (20 mm diameter, 7 mm height). A central bone defect (6 mm diameter, 5 mm depth) was inserted centrally. The bone slides were cultured for 28 days and viability was evaluated by lactate dehydrogenase and alkaline phosphatase assay, and Calcein-AM viability staining and DNA quantification. Data revealed the viability of the bone tissue over the tested time period of 28 days, and an increase in cell numbers implicating active cell proliferation processes in the sections. To analyze the bone regeneration potential of this model in combination with a bone replacement material, we injected a collagen-type 1 hydrogel into the central defect. Cellular ingrowth into the gel was evaluated by microscopy and DNA quantification at different time points demonstrating an increase of cells in the defect over time. Finally, gene expression of osteogenic markers indicated an osteoblastic phenotype of the cells in the defect. In summary, the ex vivo bone defect model remains viable and shows active bone repair processes over 28 days. Additional advantages include high reproducibility, manageable costs, and a native bone-implant interface supporting the evaluation of bone substitute materials and associated regeneration processes. Impact statement Testing of new implant materials and bone repair strategies up to date rely mainly on in vivo and in vitro investigation models providing different pros and cons. In this study we established a novel human ex vivo bone defect model with a proven vitality of at least 28 days. The model provides a native bone implant interface and is designed to monitor cell invasion into a critically sized defect filled with the potential implant material. Furthermore, associated repair processes can be documented on the cell and molecular level, including additional advantages such as high reproducibility and manageable costs.

The simultaneous analysis of mesenchymal stem cells and early osteocytes accumulation in osteoarthritic femoral head sclerotic bone.

OBJECTIVE: OA subchondral bone is a key target for therapy development. Osteocytes, the most abundant bone cell, critically regulate bone formation and resorption. Their progenitors, mesenchymal stem cells (MSCs), display altered behaviour in osteoarthritic subchondral bone. This study investigated the relationships between native osteocytes and native MSCs in osteoarthritic femoral heads. METHODS: To avoid culture manipulations, a bone treatment procedure was developed to simultaneously obtain pure osteocyte-enriched fragments and matched native CD45-CD271+ MSCs. Gene expression in osteocytes and MSCs was compared between healthy and OA bone and selected molecules were examined by immunohistochemistry in relation to OA tissue pathology. Cell sorting and standard trilineage differentiation assays were employed to test OA MSC functionality. RESULTS: Native osteocyte enrichment was confirmed histologically and by higher-level osteocyte maturation transcripts expression, compared with purified MSCs. Compared with healthy bone, native OA osteocytes expressed 9- and 4-fold more early/embedding osteocyte molecules E11 and MMP14, and 6-fold more osteoprotegerin (P<0.01). CD271+ MSCs accumulated in the regions of bone sclerosis (9-fold, P<0.0001) in close juxtaposition to trabeculae densely populated with morphologically immature E11-positive osteocytes (medians of 76% vs 15% in non-sclerotic areas, P<0.0001), and osteoblasts. Gene expression of OA MSCs indicated their bone formation bias, with retained multipotentiality following culture-expansion. CONCLUSIONS: In human late-stage OA, osteogenically-committed MSCs and adjacent immature osteocytes exhibit a marked accumulation in sclerotic areas. This hitherto unappreciated MSC-early osteocyte axis could be key to understanding bone abnormalities in OA and represents a potential target for novel therapy development in early disease.

Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells.

The cellular microenvironment is dynamic, remodeling tissues lifelong. The biomechanical properties of the extracellular matrix (ECM) influence the function and differentiation of stem cells. While conventional artificial matrices or scaffolds for tissue engineering are primarily static models presenting well-defined stiffness, they lack the responsive changes required in dynamic physiological settings. Engineering scaffolds with varying elastic moduli is possible, but often lead to stiffening and chemical crosslinking of the molecular structure with limited control over the scaffold architecture. A family of indirectly 3D printed elastomeric nanohybrid scaffolds with thermoresponsive mechanical properties that soften by reverse self-assembling at body temperature have been developed recently. The initial stiffness and subsequent stiffness relaxation of the scaffolds regulated proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages over 4 weeks, as measured by immunohistochemistry, histology, ELISA and qPCR. hBM-MSCs showed enhanced chondrogenic differentiation on softer scaffolds and osteogenic differentiation on stiffer ones, with similar relative expression to that of human femoral head tissue. Overall, stiffness relaxation favored osteogenic activity over chondrogenesis in vitro.

Hyperacute serum has markedly better regenerative efficacy than platelet-rich plasma in a human bone oxygen-glucose deprivation model.

AIM: Platelet-rich plasma (PRP) and hyperacute serum (HAS) were compared in a novel human model of ex vivo bone damage induced by oxygen-glucose deprivation (OGD). MATERIALS & METHODS: Osteoarthritic subchondral bone pieces were harvested from discarded femoral heads during hip replacement surgery and subjected to transient OGD. RESULTS: Proteome profiling revealed that PRP is more angiopoietic, whereas HAS is more antiangiopoietic in composition. However, treatment of OGD-exposed bone with multiple PRP preparations had no effect on cell counts, whereas HAS restored cell proliferation capacity and rescued viable cell number following OGD. CONCLUSION: A similar pro-proliferation effect was observed with recombinant growth factors, indicating that HAS may be an alternative agent for enhancing the regeneration of damaged bone cells.

Assessment of a closed wash system developed for processing living donor femoral heads.

NHS Blood and Transplant Tissue and Eye Services (TES) and Scottish National Blood Transfusion Services Tissues and Cells Directorate (TCD) currently bank whole, frozen femoral head bone from living donors who are undergoing primary hip replacement surgery. When required, the bone is issued to a surgeon still frozen on dry ice (- 79 °C). Consequently, the femoral head bone is not processed, is not sterilised and at the time of issue, it contains donor blood, bone marrow and associated cells. We have previously shown that, cut, shaped and washed bone from deceased donors can be processed to remove up to 99.9% of blood, bone marrow and associated cells (Eagle et al. 2015). However, cut and shaped bone is not suitable for some orthopaedic procedures and some orthopaedic surgeons do not wish to use irradiated bone; therefore in this report, a method has been developed in which whole femoral heads can be washed to remove donor blood and bone marrow components. Processing results in excess of 99% bone marrow component removal-soluble protein, haemoglobin and DNA; the procedure is performed inside a closed system, thereby eliminating the need for terminal sterilisation by irradiation. In addition, uniaxial testing demonstrated no difference in compressive strength between washed and unwashed bone. We suggest that this washed bone may be capable of improving incorporation after grafting without disturbing biomechanical properties of the graft.

Exosomes from Human Synovial-Derived Mesenchymal Stem Cells Prevent Glucocorticoid-Induced Osteonecrosis of the Femoral Head in the Rat.

Osteonecrosis of the femoral head (ONFH) represents a debilitating complication following glucocorticoid (GC)-based therapy. Synovial-derived mesenchymal stem cells (SMSCs) can exert protective effect in the animal model of GC-induced ONFH by inducing cell proliferation and preventing cell apoptosis. Recent studies indicate the transplanted cells exert therapeutic effects primarily via a paracrine mechanism and exosomes are an important paracrine factor that can be directly used as therapeutic agents for tissue engineering. Herein, we provided the first demonstration that the early treatment of exosomes secreted by human synovial-derived mesenchymal stem cells (SMSC-Exos) could prevent GC-induced ONFH in the rat model. Using a series of in vitro functional assays, we found that SMSC-Exos could be internalized into bone marrow derived stromal cells (BMSCs) and enhance their proliferation and have anti-apoptotic abilities. Finally, SMSC-Exos may be promising for preventing GC-induced ONFH.

Stratified Scaffolds for Osteochondral Tissue Engineering.

Stratified scaffolds are promising devices finding application in the field of osteochondral tissue engineering. In this scaffold type, different biomaterials are chosen to fulfill specific features required to mimic the complex osteochondral tissue interface, including cartilage, interlayer tissue, and subchondral bone. Here, the biomaterials and fabrication methods currently used to manufacture stratified multilayered scaffolds as well as cell seeding techniques for their characterization are presented.

Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.

INTRODUCTION: Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells. MATERIALS AND METHODS: MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair. RESULTS: Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs. DISCUSSION: A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.

Are CXCL13/CXCR5/FAK critical regulators of MSCs migration and differentiation?

Osteonecrosis of the femoral head is a common and challenging disease worldwide. The traditional treatments, such as core decompression procedure and joint replacement, are not satisfactory due to the limited outcome, repetitive surgery and cost. In recent years, autologous mesenchymal stem cells (MSCs) implantation into the femoral head has emerged as a promising method. The homing and differentiation of MSCs is determined by chemokines and their receptors, specific signals present in the micro-environment of the damaged tissue. CXCL13/CXCR5, highly expressed in the osteoblast and MSCs, are tissue specific and selectively migrate MSCs, thereafter triggering phosphorylation of focal adhesionkinase through mitogen-activated protein kinase pathway. Considering these characteristics, we hypothesize that CXCL13/CXCR5/FAK are critical signals in the trafficking and differentiation of MSCs.

Characterization of lipidic markers of chondrogenic differentiation using mass spectrometry imaging.

Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of cartilage defects attributable to their capacity to differentiate toward chondrocytes in the process termed chondrogenesis. The metabolism of lipids has recently been associated with the modulation of chondrogenesis and also with the development of pathologies related to cartilage degeneration. Information about the distribution and modulation of lipids during chondrogenesis could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers could be highly valuable for improving MSC-based cartilage therapies. In this work, MS imaging was used to characterize the spatial distribution of lipids in human bone marrow MSCs during the first steps of chondrogenic differentiation. The analysis of MSC micromasses at days 2 and 14 of chondrogenesis by MALDI-MSI led to the identification of 20 different lipid species, including fatty acids, sphingolipids, and phospholipids. Phosphocholine, several sphingomyelins, and phosphatidylcholines were found to increase during the undifferentiated chondrogenic stage. A particularly detected lipid profile was verified by TOF secondary ion MS. Using this technology, a higher intensity of phosphocholine-related ions was observed in the peripheral region of the micromasses collected at day 14.

Supercharging allografts with mesenchymal stem cells in the operating room during hip revision.

PURPOSE: Bone marrow derived mesenchymal stem cells (BM-MSCs) have been proposed to improve allografts used during hip revision. However, no study has reported the number of MSCs that could be associated with the allograft and the best technique to load MSCs in allografts. The optimal loading technique should combine methods to increase the initial cell density and create an appropriate environment to accelerate the efficiency of the cell-allograft constructs into clinically applicable grafts. We designed a study to evaluate the number of MSCs in an autograft femoral head considered as the gold standard and to determine the best operating room procedure for loading in allograft with MSCs to approach the same number as in an autograft femoral head. Therefore this study explored a potential of charging whole femoral head allografts with autologous MSCs from iliac crest aspirate for hip revision procedures. METHODS: First, the study evaluated the total number of mesenchymal stem cells (MSCs) in 1 cc of an average autograft femoral head; this number then serves as a target for loading allografts, in order to achieve the same density of MSCs. For the loading technique itself, several questions were asked and hence several options were investigated. For example, is it better to load the whole allograft or break it up into several fragments? Which way of injecting works best for the whole femoral head allograft (through cartilage or femoral neck)? How concentrated (in terms of MSCs) should the injected iliac crest marrow be? Bone marrow for injection in allografts was obtained from residual marrow from patients undergoing surgical procedures with concentrated bone marrow. With this bone marrow (with and without concentration) we tested different techniques (injection and soaking) to load stem cells in allografts of different sizes: bulk allografts, pieces or blocks (8 or 1 cm(3) blocks) and morselized fragments (from 125 to 8 mm(3)) or particules (1 mm(3)). We also evaluated the release of MSCs from fragments of autografts and allografts loaded with MSCs in cultured medium. RESULTS: The femoral head autografts contained a lower concentration of MSCs than the iliac crests of the same patient. However, in absence of concentration, with bone marrow aspirated from the iliac crest, we were not able to load in the femoral head allograft the same number of MSCs as the number present in an autograft. The loaded volume of bone marrow (and the corresponding number of MSCs) depended on the technique (injecting, soaking) as well as on the volume and shape of the allografts. The seeding efficiency of loading MSCs in allografts increased with the concentration of MSCs in the bone marrow. With concentrated bone marrow, supercharging the allograft with MSCs (as compared with an autograft) was possible in the operating room, and the number of MSCs supercharged in allografts was predictable. CONCLUSIONS: The loaded volume of bone marrow depended on the technique (injecting, soaking) as well as on the volume and shape of the allografts. With concentrated bone marrow, the allograft could be charged with a similar or higher number of MSCs than the number present in a femoral head autograft.

Morphological and functional characterization of femoral head drilling-derived mesenchymal stem cells.

Adult mesenchymal stem cells (MSCs) were primary identified as bone marrow-derived cells, fibroblast-like morphology, and adherent to plastic surfaces of in vitro culture plate. Their identification criteria evolved in time to a well-established panel of markers (expression of CD73, CD90, and CD105) and functional characteristics (adipogenic, osteogenic, and chondrogenic trilineage differentiation ability), which can be applied to adult mesenchymal stem cells obtained from other tissue sources. We tried to assess the potential stemness of femoral head drilling-derived cells as a new source of mesenchymal stem cells (FH-MSCs). For this purpose, we used the morphological and ultrastructural characteristics defined by scanning and transmission electron microscopy and spindle-shape cellular body, fibroblast-like, with few thick elongations (lamellipodia) and numerous fine, thin cytoplasmic projections (filopodia) that extend beyond the edge of lamellipodia. Immunophenotypical analysis was performed by flow cytometry and immunocytochemical methods and we showed that FH-MSCs share the characteristic markers of MSCs, expressing CD73, CD90, CD105, and being positive for vimentin, and c-kit (CD117). Proliferation rate of these cells was moderate, as revealed by Ki67 immunostaining. Regarding the functional characteristics of FH-MSCs, after appropriate time of induction in specific culture media, the cells were able to prove their trilineage potential and differentiated towards adipocytic, osteogenic, and chondrogenic lineage, as revealed by immunofluorescent staining. We may conclude that femoral head drilling-derived cells can be used as a novel source of stem cells, and employed in diverse clinical settings.

Rosuvastatin inhibits spontaneous and IL-1ß-induced interleukin-6 production from human cultured osteoblastic cells.

OBJECTIVE: Experimental and clinical data suggest that statins may protect bone by inhibiting bone resorption and/or stimulating bone formation. Interleukin-6 (IL-6) is produced by osteoblasts, and potently stimulates osteoclast activation playing a key role in normal bone resorption as well as in post-menopausal and inflammation-driven osteoporosis. Although statins inhibit IL-6 production from different cell types, currently no data exist on osteoblasts. The aim of the study was to evaluate the effect of rosuvastatin on IL-6 production by human osteoblasts. METHODS: Osteoblasts from osteoarthritic patients were incubated with rosuvastatin (0.1-10 µmol/L)±IL-1ß, and IL-6 production was evaluated as cytokine concentration in the culture medium (ELISA), as well as mRNA expression in the cells (qPCR). Putative intracellular mechanisms of the drug, such as blocking HMG-CoA-reductase, and interference in the prenylation process were investigated by the addition of mevalonate and isoprenoids. The effect of rosuvastatin±IL-1ß on the anti-resorptive molecule osteoprotegerin (OPG) was also assessed (ELISA). RESULTS: Rosuvastatin significantly reduced IL-6 levels in the osteoblast culture medium, both in unstimulated and IL-1ß-stimulated cells. This effect was reversed by mevalonate or geranylgeraniol, but not farnesol. Moreover, the drug decreased both spontaneous and IL-1ß-induced IL-6 mRNA expression in osteoblasts. Conversely, rosuvastatin did not affect OPG levels in the culture medium. CONCLUSION: Our results show that rosuvastatin decreases IL-6 production by osteoblasts, thereby suggesting a possible inhibiting activity on osteoclast function in an indirect way. These data may provide further rationale for employing rosuvastatin to beneficially affect bone metabolism in post-menopausal women and possibly in inflammation-driven osteoporosis.

Monosodium iodoacetate induces apoptosis via the mitochondrial pathway involving ROS production and caspase activation in rat chondrocytes in vitro.

Monosodium iodoacetate (MIA) is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase activity, and causes dose-dependent cartilage degradation resembling the pathological changes of human osteoarthritis (OA). In this study, we assessed the apoptosis induced by MIA and clarified the underlying mechanisms using the primary rat chondrocytes. The apoptosis of primary rat chondrocytes was analyzed by flow cytometry. The levels of mitochondrial membrane potential (ΔΨm) were evaluated using fluorescence spectrophotometer. The production of reactive oxygen species (ROS) was determined by fluorescence spectrophotometer. Apoptosis-related protein cytochrome c and procaspase-3 expressions were examined by Western blotting. We found that MIA treatment induces apoptosis in chondrocytes, as confirmed by increases in the percent of apoptotic cells, up-regulation of cytochrome c and caspase-3 protein levels. Treatment with MIA increases ROS production and decreases the levels of ΔΨm. The antioxidant, N-acetylcysteine (NAC), significantly prevented the production of ROS, the reduction of ΔΨm, the release of cytochrome c and the activation of caspase-3. Further, NAC completely protected the cells from MIA-induced apoptosis. Together these observations suggest that the mechanisms of MIA-induced apoptosis are primarily via ROS production and mitochondria-mediated caspase-3 activation in primary rat chondrocytes.