Qiming Fan

In vitro responses of human bone marrow stromal cells to a fluoridated hydroxyapatite coated biodegradable Mg—Zn alloy

Bone-like fluoridated hydroxyapatite (FHA) coatings were prepared on Mg-6 wt.%Zn substrates using electrochemical method. Human bone marrow stromal cells (hBMSCs) were utilized to investigate the cellular biocompatibility of Mg-6 wt.%Zn alloy after surface modification. The adhesion of hBMSCs was evaluated using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The proliferation of the cells was also measured by carrying out the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. And the alkaline phosphatase activity (ALP) was assessed to evaluate the early stage of differentiation. Lastly, reverse transcription-polymerase chain reaction (RT-PCR) test was taken. It was found that the hBMSCs displayed better cell functions on the bioactive FHA coated alloy, compared to the bare Mg-6 wt.%Zn alloy. The in vitro results indicated that the bioactive FHA coating can improve the interfacial bioactivity of Mg-6 wt.%Zn substrate, specifically, both on biodegradation behavior control and good cellular proliferation and differentiation.

Human mesenchymal stem cells (hMSCs) target osteosarcoma and promote its growth and pulmonary metastasis.

In an effort to study the interaction between MSCs and osteosarcoma, we established an animal model of primary osteosarcoma in nude mice using Saos-2 cells. hMSCs, labeled with adv-GFP, were injected through the caudal vein. We observed that exogenous hMSCs targeted the osteosarcoma site and promoted its growth and pulmonary metastasis in vivo. To elucidate the underlying mechanisms, we employed transwell, neutralization antibody and MTT assays in vitro. hMSCs migrated toward the conditioned medium from Saos-2 cells, and SDF-1 was involved in this migration. Likewise, Saos-2 cells migrated toward the conditioned medium from hMSCs and CCL5 played an important role in this migration. Furthermore, proliferation of Saos-2 cells was enhanced by the conditioned medium from hMSCs and CCL5 was at least partly responsible for this enhancement.

STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma.

We previously demonstrated that human mesenchymal stem cells (MSCs) promote the growth of osteosarcoma in the bone microenvironment. The aim of the present study was to further determine the effect of IL-6/STAT3 signaling on the progression of osteosarcoma. First, conditioned medium from MSCs was used to stimulate the growth of osteosarcoma cells (Saos-2) in vitro. We found that STAT3 was activated and that the activation could be blocked by an IL-6-neutralizing antibody. The inhibition of STAT3 in Saos-2 cells by siRNA or AG490 decreased cell proliferation, migration and invasion, down-regulated the mRNA expression of Cyclin D, Bcl-xL and Survivin and enhanced the apoptotic response. Furthermore, a nude mouse osteosarcoma model was established by injecting luciferase-labeled Saos-2 cells into the tibia, and the effect of STAT3 on tumor growth was determined by treating the mice with AG490. In vivo bioluminescence images showed that tumor growth was dramatically reduced in the AG490 group. In addition, STAT3 inhibition decreased the lung metastasis rate and prolonged the survival of these mice. After treatment with AG490, the protein levels of IL-6, p-STAT3 and PCNA were decreased, and the level of apoptosis in the tumor was increased. Altogether, these data indicate that MSCs in the bone microenvironment might promote the progression of osteosarcoma and protect tumor cells from drug-induced apoptosis through IL-6/STAT3 signaling.

The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells

Although Sox9 is essential for chondrogenic differentiation and matrix production, its application in cartilage tissue engineering has been rarely reported. In this study, the chondrogenic effect of Sox9 on bone marrow mesenchymal stem cells (BMSCs) in vitro and its application in articular cartilage repair in vivo were evaluated. Rabbit BMSCs were transduced with adenoviral vector containing Sox9. Toluidine blue, safranin O staining and real-time PCR were performed to check chondrogenic differentiation. The results showed that Sox9 could induce chondrogenesis of BMSCs both in monolayer and on PGA scaffold effectively. The rabbit model with full-thickness cartilage defects was established and then repaired by PGA scaffold and rabbit BMSCs with or without Sox9 transduction. HE, safranin O staining and immunohistochemistry were used to assess the repair of defects by the complex. Better repair, including more newly-formed cartilage tissue and hyaline cartilage-specific extracellular matrix and greater expression of several chondrogenesis marker genes were observed in PGA scaffold and BMSCs with Sox9 transduction, compared to that without transduction. Our findings defined the important role of Sox9 in the repair of cartilage defects in vivo and provided evidence that Sox9 had the potential and advantage in the application of tissue engineering.

The effect of metallic magnesium degradation products on osteoclast-induced osteolysis and attenuation of NF-κB and NFATc1 signaling.

Wear particle-induced aseptic prosthetic loosening is one of the most common reasons for total joint arthroplasty (TJA). Extensive bone destruction (osteolysis) by osteoclasts plays an important role in wear particle-induced peri-implant loosening. Thus, strategies for inhibiting osteoclast function may have therapeutic benefit for prosthetic loosening. Here, we mimicked the process of magnesium (Mg) degradation in vivo and obtained Mg leach liquor (MLL) by immersing pure Mg in culture medium. For the first time, we demonstrated that MLL suppresses osteoclast formation, polarization, and osteoclast bone resorption in vitro. An in vivo assay demonstrated that MLL attenuates wear particle-induced osteolysis. Furthermore, we found that MLL significantly inhibits nuclear factor-κB (NF-κB) activation by retarding inhibitor-κB degradation and subsequent NF-κB nuclear translocation. We also found that MLL attenuates the expression of NFATc1 at both the protein and mRNA levels. These results demonstrate that MLL has anti-osteoclast activity in vitro and prevents wear particle-induced osteolysis in vivo. Collectively, our study suggests that metallic magnesium, one of the orthopedic implants with superior properties, has significant potential for the treatment of osteolysis-related diseases caused by excessive osteoclast formation and function.

Human mesenchymal stem cells promote growth of osteosarcoma: Involvement of interleukin‐6 in the interaction between human mesenchymal stem cells and Saos‐2

Our previous study showed that exogenous human mesenchymal stem cells (hMSCs) targeted established osteosarcoma and promoted its growth and pulmonary metastasis in vivo. As a follow‐up, the present study aimed to investigate how hMSCs would interact with Saos‐2 through autocrine/paracrine communication. The results showed that co‐injection of hMSCs with Saos‐2 into the proximal tibia of nude mice could promote tumor growth and progression. In vitro, the proliferation of Saos‐2 and hMSCs was promoted by each other’s conditioned medium, in which interleukin‐6 (IL‐6) played an important role. Osteogenic differentiation of hMSCs could be inhibited by conditioned medium of Saos‐2, in which IL‐6 was also involved. Furthermore, decreased IL‐6 secretion by hMSCs during its osteogenesis and increased IL‐6 secretion in response to conditioned medium of Saos‐2 were observed. Based on these data, we suggest that there was a positive feedback loop of IL‐6 in the interaction between hMSCs and Saos‐2. (Cancer Sci 2010; 101: 2554–2560)

The use of autologous enriched bone marrow MSCs to enhance osteoporotic bone defect repair in long-term estrogen deficient goats

Bone defects are common in elderly patients suffering from osteoporosis. Current methods of bone defect treatment for osteoporosis are not always satisfactory. In this study, we demonstrated that bone marrow mesenchymal stem cells (MSCs) harvested from goats with long-term estrogen deficiencies exhibited a lower proliferation rate and decreased osteogenic capacity, which are critical obstacles for bone defect repair in the elderly. However, by combining autologous enriched bone marrow mesenchymal stem cells with porous β-TCP, we successfully repaired critical-sized bone defects in the medial femoral condyle of the osteoporotic goats. Both micro-CT images and histomorphometry analysis illustrated improved bone formation following the enriched MSC therapy. Thus, we proposed autologous enriched bone marrow mesenchymal stem cells as a quick, safe therapeutic strategy to treat osteoporotic bone defects.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway.

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells.

Hypericin suppresses osteoclast formation and wear particle-induced osteolysis via modulating ERK signalling pathway

Osteoclast-induced bone resorption and wear-particle-induced osteolysis leads to prosthetic loosening, one of the most common causes of joint implant failure, resulting in revision surgery. Thus, inhibition of osteoclastic bone resorption, which further prevents wear particle-induced osteolysis, is a potential treatment strategy for prosthetic loosening. Here, we examined the therapeutic effect of hypericin (HP), which was photosensitive, on osteoclastogenesis and wear particle-induced osteolysis in the absence of visible light. HP inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) and RAW264.7 cell line without any evidence of cytotoxicity. The bone-resorbing activity of mature osteoclasts was significantly inhibited by HP. As HP has been previously reported to inhibit signalling pathway such as ERK and NF-κB in other cells, which is also important in osteoclast differentiation. We thus examined the molecular mechanism and showed that HP significantly inhibited the ERK/mitogen-activated protein kinase (MAPK) signalling pathway without affecting nuclear factor kappaB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 signalling in RANKL-stimulated BMMs. Further in vivo studies revealed HP attenuated osteoclast formation and subsequently prevented wear particle-induced bone erosion. Taken together, the results suggest that HP inhibits RANKL-mediated osteoclastogenesis via affecting ERK signalling in vitro and suppresses wear particle-induced osteolysis in vivo. We therefore conclude that HP may be an innovative and safe alternative treatment for osteoclast-related prosthetic loosening.

Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades

Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases.