Hongxing Jiang

Osteogenic differentiation of human mesenchymal stem cells cultured with dexamethasone, vitamin D3, basic fibroblast growth factor, and bone morphogenetic protein-2.

Purpose: Human mesenchymal stem cells (hMSCs) are pursued for cell-based therapies of bone defects. Successful use of hMSCs will require them to be osteogenically differentiated before transplantation. This study was intended to determine the optimal combination(s) of supplements needed for inducing osteogenesis in hMSCs. Methods: The hMSCs were cultured with combinations of β-glycerophosphate, dexamethasone (Dex), vitamin D3 (Vit-D3), basic fibroblast growth factor (bFGF), and bone morphogenetic protein-2 (BMP-2) to assess cell growth and osteogenesis. Osteogenic responses of the supplements were evaluated by alkaline phosphatase (ALP) activity, mineralization, and gene expression of ALP, Runx2, bone sialoprotein, and osteonectin. Adipogenesis was characterized based on Oil Red O staining, gene expression of peroxisome proliferator-activated receptor γ2, and adipocyte protein-2. Results: Dex was found to be essential for mineralization of hMSCs. Cultures treated with Dex (100 nM), Vit-D3 (10/50 nM), and BMP-2 (500 ng/mL) demonstrated maximal calcification and up-regulation of ALP and bone sialoprotein expression. However, adipogenesis was up-regulated in parallel with osteogenesis in these cultures, as evident by the presence of lipid droplets and significant up-regulation of peroxisome proliferator-activated receptor γ2 and adipocyte protein-2 expression. An optimal condition was obtained at Dex (10 nM) and BMP-2 (500 ng/mL) for mineralization without increasing adipogenesis-related markers. The bFGF mitigated osteogenesis and enhanced adipogenesis. Vit-D3 appears essential for calcification only in the presence of bFGF. Conclusion: Treatment of hMSCs with appropriate supplements at optimal doses results in robust osteogenic differentiation with minimal adipogenesis. These findings could be used in the cultivation of hMSCs for cell-based strategies for bone regeneration.

MRI characteristics of the neurocentral synchondrosis.

Abstract Background and objectives. The neurocentral synchondrosis (NCS) is a cartilaginous growth plate that since the early 1900s has been implicated as a potential cause of adolescent idiopathic scoliosis (AIS). Previous studies have focused only on the closure age without characterizing normal NCS development. Using MRI, the normal development of the NCS image can be characterized, and the stages preceding the disappearance of this image can be specified. Methods. A total of 405 NCSs were visualized in 11 normal pediatric patients using T1 and T2 transverse and sagittal MRI views. The images were correlated and the variety of images recorded to categorize the NCS into developmental stages. Results. The development of the NCS was categorized into five developmental stages. The image of the NCS became absent in a specific pattern along the vertebral column, first in the cervical region (age 6), then in the lumbar region (age 12), and finally in the thoracic region (age 14). Conclusion. The normal development of the NCS at the level of individual vertebrae and also along the vertebral column as a whole was determined using MRI. These patterns of development are valuable and necessary to evaluate the role of the NCS in pathological conditions.

Noggin suppression decreases BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells In Vitro†

Numerous studies with rodent cells and animal models indicate that noggin inhibits osteogenesis by antagonizing bone morphogenetic proteins (BMPs); however, the effect of noggin on osteogenesis of human cells remains ambiguous. This study aims to examine the effects of noggin suppression on viability and BMP‐2‐induced osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells (MSCs) in vitro. Noggin expression in human MSCs was suppressed by noggin‐specific small interfering RNA (siRNA), and viability of human MSCs was determined by measuring the mitochondrial dehydrogenase activity, cellular DNA content and protein amount. The BMP‐2‐induced osteogenic differentiation of human MSCs was assessed by analyzing the expression levels of several osteoblastic genes, enzymatic alkaline phosphatase (ALP) activity and calcification. Our study showed that noggin suppression significantly decreased human MSC metabolism and DNA content on Days 3 and 6, and decreased total protein amount on Day 14. Noggin suppression also reduced the expression levels of osteoblastic genes, ALP, integrin‐binding sialoprotein (IBSP), muscle segment homeobox gene (MSX2), osteocalcin (OC), osteopontin (OPN), and runt‐related transcription factor‐2 (RUNX2). Significantly decreased enzymatic ALP activity in noggin‐suppressed group was evident. Moreover, noggin suppression decreased calcium deposits by BMP‐2‐induced osteoblasts. Collectively, this study showed that noggin suppression decreased viability and BMP‐2‐induced osteogenic differentiation of human MSCs, which suggests that noggin is stimulatory to osteogenesis of human MSCs. J. Cell. Biochem. 113: 3672–3680, 2012.

Targeting CXCR4/SDF-1 axis by lipopolymer complexes of siRNA in acute myeloid leukemia

In spite of high complete remission rates in Acute Myeloid Leukemia (AML), little progress has been made in the long-term survival of relapsing AML patients, urging for the development of novel therapies. The CXCR4/SDF-1 axis is a potential therapeutic target in AML to reduce the enhanced survival and proliferation of leukemic cells, with current drug development efforts focusing on antagonists and blocking antibodies. The RNAi technology mediated by siRNA is a promising alternative; however, further development of clinically relevant siRNA carriers is needed since siRNA on its own is an incompetent silencing agent. Here, we report on lipid-substituted polymeric carriers for siRNA delivery to AML cells, specifically targeting CXCR4. Our results demonstrate an effective suppression of CXCR4 protein with the polymeric siRNA delivery in AML THP-1 cells. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), decreased THP-1 cell numbers due to reduced cell proliferation. The reduced proliferation was also observed in the presence of human bone marrow stromal cells (hBMSC), suggesting that our approach would be effective in the protective bone marrow microenvironment. The combination of CXCR4 silencing and cytarabine treatment resulted in more effective cytotoxicity when the cells were co-incubated with hBMSC. We observed a decrease in the toxicity of the lipopolymer/siRNA complexes when THP-1 cells were treated in the presence of hBMSC but this effect did not negatively affect CXCR4 silencing. In addition, siRNA delivery to mononuclear cells derived from AML patients led to significant CXCR4 silencing in 2 out of 5 samples, providing a proof-of-concept for clinical translation. We conclude that decreasing CXCR4 expression via lipopolymer/siRNA complexes is a promising option for AML therapy and could provide an effective alternative to current CXCR4 inhibition strategies.

Macrophages Inhibit Migration, Metabolic Activity and Osteogenic Differentiation of Human Mesenchymal Stem Cells in vitro

To better elucidate the role of macrophages in bone morphogenetic protein (BMP)-induced bone repair, this study evaluated the effects of macrophages on the migration, metabolic activity and BMP-2-induced osteogenic differentiation of human mesenchymal stem cells (hMSCs). Human monocytes were induced into a macrophage phenotype, and the conditioned media (CM) from undifferentiated monocytes and differentiated macrophages were collected for treatment of hMSCs. Expression levels of osteoblastic marker genes, alkaline phosphatase (ALP) activity and mineral deposition were assessed. The migration of hMSCs was significantly decreased after treatment with the macrophage CM (but not monocyte CM), in a dose-dependent manner. Significant inhibition of hMSC metabolism was observed on days 3 and 7 after treatment with the macrophage CM. The osteoblastic marker genes analyzed (ALP, bone sialoprotein, osteocalcin and runt-related transcription factor-2) after exposure of hMSCs to BMP-2 were all significantly downregulated in cells treated with the macrophage CM. The hMSCs treated with macrophage CM showed significantly decreased enzymatic activity of ALP and calcium content compared with those treated with monocyte CM or basal medium. High levels of interleukin-1β and tumor necrosis factor-α found in macrophage CM may mediate these observed effects on hMSCs. We conclude that macrophage CM suppressed the BMP-2-induced osteogenic differentiation of hMSCs, suggesting that macrophages might contribute to decreased osteogenic effects of BMPs in a clinical setting.

Modification of human BMSC with nanoparticles of polymeric biomaterials and plasmid DNA for BMP-2 secretion

BACKGROUND Genetic modification of human bone marrow stem cells (hBMSCs) before administration to a patient is emerging as a viable approach to creating tailored cells that perform effectively in a clinical setting. To this end, safe delivery systems are needed that can package therapeutic genes into nanoparticles for cellular delivery. METHODS We evaluated different plasmids on gene expression and compared the effective plasmids directly in hBMSCs. Then, we evaluated the transfection efficiencies of the polymeric carriers linoleic acid-substituted polyethylenimine (PEI-LA), polyethylenimine (PEI)-25, and PEI-2 using flow cytometry. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to compare the toxicity of PEI-LA and PEI-25 on hBMSCs. We further assessed bone morphogenetic protein-2 (BMP-2) secretion and the osteogenic activity of hBMSCs transfected with the polymeric (PEI-LA and PEI-25) gWIZ-BMP-2 complex. RESULTS Unlike the transformed cells that gave robust (>50%) transfection, only a few percent (<10%) of hBMSCs was transfected by the developed nanoparticles in culture. The plasmid DNA design was critical for expression of the transgene product, with the choice of the right promoter clearly enhancing the efficiency of transgene expression. Using the in-house designed PEI-LA, hBMSCs secreted BMP-2 in culture (~4 ng BMP-2/10(6) cells/d), which indicates the feasibility of using PEI-LA as a delivery system. Furthermore, we demonstrated an increased osteogenic activity in vitro for hBMSCs transfected with the PEI-LA containing the BMP-2 expression system. CONCLUSIONS These results provide encouraging evidence for the potential use of a low toxic PEI-LA to genetically modify hBMSC.

A comparison of the innervation characteristics of the lateral spinal ligaments between normal subjects and patients with adolescent idiopathic scoliosis.

Evidence is rapidly accumulating to suggest that general proprioceptive dysfunction might be a major contributing factor in the development of adolescent idiopathic scoliosis (AIS). The innervation of appropriate ligaments which has been shown to be involved in proprioceptive feedback mechanisms, has also been suggested to play a part in this sensory dysfunction. Accordingly, this study compared the innervation characteristics of lateral spinal ligaments from patients with AIS to similar measurements from control subjects. Using an antibody to neurofilament protein, Ruffini corpuscles, small and large nerve bundles, and free nerve endings were identified and their numbers and distribution patterns compared. In the control group, the innervation was found to be symmetrical between left and right sides but was more concentrated in the ventral portion of each ligament. No apparent morphological defect of the innervation was found in the lateral spinal ligaments of the scoliosis patients but the innervation densities of Ruffini corpuscles, single nerve fibres and total neural elements were significantly lower (p<0.01) than those found in normal subjects. These results suggest a possible mechanism for the production of AIS and warrant further study.