Huifang Zhou

Effects of a miR-31, Runx2, and Satb2 Regulatory Loop on the Osteogenic Differentiation of Bone Mesenchymal Stem Cells

Recently, a cohort of miRNAs, including miR-31, was reported to be downregulated during osteogenic induction by miR microarray analysis. It remains unclear how changes in miR-31 expression collaborate with bone transcription factors to activate the biological pathways that regulate the differentiation of bone mesenchymal stem cells (BMSCs). Here the effects of miR-31, Runx2, and Satb2 on the osteogenic differentiation of BMSCs were investigated using mimics and inhibitors of miR-31, small interfering RNA for knockdown of Runx2 and plasmids for overexpression of Runx2. Our results showed that miR-31 expression decreased progressively in BMSC cultures during differentiation. Inhibition of miR-31 dramatically increased the alkaline phosphatase activity and mineralization in BMSC cultures. Additionally, miR-31 diminished the levels of the Satb2 protein without significantly affecting Satb2 mRNA levels, and Runx2 directly repressed miR-31 expression. Overexpression of miR-31 significantly reduced expression of the osteogenic transcription factors OPN, BSP, OSX, and OCN, but not Runx2. Furthermore, the high expression of miR-31 in BMSCs cultured in the proliferation medium repressed Satb2 protein levels, which may contribute to the maintenance of BMSCs in an undifferentiated state. In conclusion, our results suggest that a Runx2, Satb2, and miR-31 regulatory mechanism may play an important role in inducing BMSC osteogenic differentiation. The results of this study provide us with a better understanding of the molecular mechanisms that govern the BMSC fate.

Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration*

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

Recent advances in bioprinting techniques: approaches, applications and future prospects.

Bioprinting technology shows potential in tissue engineering for the fabrication of scaffolds, cells, tissues and organs reproducibly and with high accuracy. Bioprinting technologies are mainly divided into three categories, inkjet-based bioprinting, pressure-assisted bioprinting and laser-assisted bioprinting, based on their underlying printing principles. These various printing technologies have their advantages and limitations. Bioprinting utilizes biomaterials, cells or cell factors as a “bioink” to fabricate prospective tissue structures. Biomaterial parameters such as biocompatibility, cell viability and the cellular microenvironment strongly influence the printed product. Various printing technologies have been investigated, and great progress has been made in printing various types of tissue, including vasculature, heart, bone, cartilage, skin and liver. This review introduces basic principles and key aspects of some frequently used printing technologies. We focus on recent advances in three-dimensional printing applications, current challenges and future directions.

Late reconstruction of the complex orbital fractures with computer-aided design and computer-aided manufacturing technique.

Background: To construct three-dimensional (3D) imaging and computer generated models of complex orbital fractures, and develop a Computer-Aided Design/Computer-Aided Manufacture (CAD/CAM) system to help improve the surgical planning of complex orbital fracture and promote its outcome. Methods: A prospective study was carried out on 17 patients with unilateral complex orbital fractures from Mar 2003 to Mar 2006 at the Shanghai ninth peoples hospital. The utilization of a CAD/CAM technique based on Helical computer tomography data, with stereolithographical (SLA) modelling as intermediate step, enabled surgeons to plan for the surgical progress of osteotomy, movement, reposition, fixation and material implanting. Orbital volume was calculated pre and post-operatively. Orbital fracture reconstruction and globe repositioning was performed and followed up 3-9 months post-treatment. Ocular function and aesthetic deformities such as enophthalmos, diplopia and extraocular motility problems were accessed. The data was processed with SAS 6.17 statistical software. Results: 17 patients with complex orbital fractures underwent successful orbital fracture reconstruction surgery. The deformities of orbit, medial canthus, nose, zygomata, maxillary and frontal bone were well corrected. The volume of reconstructed orbit was approximately symmetrical with respect to the contralateral orbit. Enophthalmos was corrected and diplopia, extraocular movement were improved. Conclusions: CAD/CAM system enables the surgeon to predict reconstructive surgical steps before the operation, and can help to improve the outcome of surgery. This technique may be proved as one of the most useful clinical tools for orbital surgery.

Effects of miR-31 on the osteogenesis of human mesenchymal stem cells.

Exploring the molecular mechanisms that regulate the osteogenesis of human mesenchymal stem cells (hMSCs) will bring us more efficient methods for improving the treatment of bone-related diseases. In this study, we analyzed the effects of miR-31 on the osteogenesis of hMSCs. The overexpression of miR-31 repressed the osteogenesis of hMSCs, whereas the downregulation enhanced this process. SATB2 was testified to be a direct target of miR-31, and its effects on the osteogenesis were also described. Most importantly, the knockdown of SATB2 attenuated miR-31s osteogenic effects. Taken together, our findings suggest that miR-31 regulates the osteogenesis of hMSCs by targeting SATB2.

Conditioned Medium from Bone Marrow Mesenchymal Stem Cells Transiently Retards Osteoblast Differentiation by Downregulating Runx2

Mesenchymal stem cells (MSCs) are attractive candidates for cell therapy and regenerative medicine because of their potential for proliferation and multilineage differentiation. MSCs secrete various cytokines, acting as trophic mediators to regulate neighboring cells. Osteoblasts are the cells directly responsible for forming new bone, and they are the final target of many osteogenic regulators. However, the induction effect of MSCs on osteoblasts is still unknown. In this study, we isolated osteoblasts from rat calvariae and investigated their proliferation and differentiation under induction of varied concentrations of MSC-conditioned medium (MSC-CM). Cells in the MSC-CM groups showed a reduction in cell proliferation at 3–6 days, and a decrease in the expression of osteocalcin and osteopontin at 3 days, with low levels of alkaline phosphatase activity. The expression of osteogenic markers went back to normal at 7 days. In order to evaluate the molecular mechanisms underlying this suppression, levels of two osteoblastic transcription factors, runt-related transcription factor 2 (Runx2) and osterix (Osx), were detected at both mRNA and protein levels. The results indicated that MSC-CM significantly inhibited Runx2 expression in a concentration-dependent manner. However, the effect was not due to the inhibition of Osx, for Osx was not significantly altered. This work demonstrates that MSCs may suppress osteoblast proliferation and transiently retard osteoblast differentiation by downregulating Runx2. These results highlight the need to take into account the paracrine effect of MSCs when using them in regenerative medicine for the repair of bone defects.

Insulin-like growth factor 1 promotes the proliferation and adipogenesis of orbital adipose-derived stromal cells in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is characterised by increased volume of the orbital contents involving adipose tissue, but the factors responsible for stimulation of orbital adipogenesis remain uncertain. Previous studies have shown that insulin-like growth factor 1 (IGF-1) is increased in the orbital fatty connective tissues of patients with TAO. The present study was conducted to investigate the effects of IGF-1 on orbital adipose-derived stromal cells (OADSCs) derived from TAO patients and to identify the signalling mechanisms involved. Our results showed that IGF-1 significantly promoted the cell proliferation and lipid accumulation of TAO OADSCs. The mRNA expression of adipogenic markers (adiponectin, leptin, adipocyte fatty acid binding protein [AP2] and fatty acid synthase [FAS]) was increased in TAO cultures treated with IGF-1. Further research demonstrated that the protein levels of peroxisome proliferator-activated receptor-γ (PPARγ) were up-regulated when OADSCs were treated with IGF-1. We also found that the inhibition of either IGF-1 receptor (IGF-1R) or phosphoinositide 3-kinase (PI3K) activity decreased the levels of IGF-1-stimulated mRNA encoding adiponectin, leptin, AP2, and FAS, as well as PPARγ protein levels. Moreover, the expression of phosphorylated Akt (p-Akt) protein in TAO cells was up-regulated by IGF-1, while a specific PI3K inhibitor (LY294002) or an antibody of IGF-1R blocked this effect. These results indicate that IGF-1 is a pro-proliferative and pro-adipogenic factor in TAO OADSCs. IGF-1 enhances the adipogenesis of TAO OADSCs by up-regulation of PPARγ via the activation of the IGF-1R and PI3K pathways, suggesting that the blocking of IGF-1R or inhibition of PI3K signalling might be a potential novel therapeutic approach to TAO.

An epidemiological study of sleep quality in adolescents in South China: a school-based study.

BACKGROUND This study explored the prevalence of disturbed sleep and investigated its distribution characteristics and associated factors in adolescents in South China. METHODS Junior middle school and senior high school students (n = 1221) were recruited from schools in Shanghai, China. Students completed a questionnaire using the Pittsburgh Sleep Quality Index and factors associated with disturbed sleep. RESULTS The prevalence of a tendency towards poor sleep was 34.32% [95% confidence interval (CI): 31.66-36.98] with no significant difference between genders. This tendency increased with age, yielding a significant group effect (P < 0.01). In middle school and high school, the propensity towards poor sleep was 31.34% (95% CI: 28.29-34.39) and 42.22% (95% CI: 36.92-47.52) respectively. The factors associated with poor sleep were more television viewing during weekdays [odds ratio (OR): 1.56, CI: 1.36-1.71], more frequent computer/Internet use (OR: 1.25, CI: 1.08-1.39), earlier school starting time (OR: 1.12, CI: 1.07-1.28), and more time on homework during weekdays (OR: 1.78, CI: 1.51-1.98) and weekends (OR: 1.35, CI: 1.21-1.52) CONCLUSIONS A tendency towards poor sleep is common in adolescents in South China and its incidence increases with age. The factors associated with this phenomenon indicate that poor sleep in adolescents could be improved, at least partly, by reducing the use of visual technologies and by changing school timetables.

Characterization of human ethmoid sinus mucosa derived mesenchymal stem cells (hESMSCs) and the application of hESMSCs cell sheets in bone regeneration

Mesenchymal stem cells (MSCs) have been extensively applied in the field of tissue regeneration. MSCs derived from various tissues exhibit different characteristics. In this study, a cluster of cells were isolated from human ethmoid sinus mucosa membrane and termed as hESMSCs. hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation. In particular, hESMSCs displayed strong osteogenic differentiation potential, and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Next, hESMSCs were prepared into a cell sheet and combined with a PSeD scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats, which showed excellent reparative effects. Additionally, ELISA assays revealed that secreted cytokines, such as BMP-2, BMP-4 and bFGF, were higher in the hESMSCs conditioned medium, and immunohistochemistry validated that hESMSCs cell sheet promoted the expression of BMP signaling downstream genes in newly formed bone. In conclusion, hESMSCs were demonstrated to be a class of mesenchymal stem cells that possessed high self-renewal capacity along with strong osteogenic potential, and the cell sheet of hESMSCs could remarkably promote new bone regeneration, indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone regeneration.

In Vivo Efficacy of Bone Marrow Stromal Cells Coated with Beta-Tricalcium Phosphate for the Reconstruction of Orbital Defects in Canines

PURPOSE To repair the segmental orbital rim defects of dogs with three-dimensional (3D) tissue-engineered constructs derived from culturing autogenous bone marrow stromal cells (BMSCs) on β-tricalcium phosphate (β-TCP) scaffolds. METHODS A 25-mm segmental defect on the canine inferior orbital rim was created. BMSCs were isolated and osteogenically induced in vitro, then were seeded onto 3D β-TCP scaffolds and implanted to repair the orbital defects after 5 days of cultivation. The group of noninduced BMSC/β-TCP, β-TCP alone, and the normal inferior orbital rim were set as controls. The orbits of all groups had spiral computed tomography (CT) scans 1, 4, 8, and 12 weeks after surgery. Gross examination, bone density, microCT, and histologic measurements were performed 12 weeks after surgery. The results were analyzed to evaluate the extent of bone repair. RESULTS Twelve weeks after surgery, CT examination revealed good inferior orbital rim recovery in the induced BMSC/β-TCP group, and the bone density was 0.30 ± 0.03 g/cm(2) with no dominant variance, compared with the normal control (P > 0.05). MicroCT and histologic examination confirmed that the implantations led to good repair of the defects. Pore-like spongy bone surrounded the implants through the section plane, with some residue remaining in the center. In contrast, the noninduced BMSC/β-TCP implants were not fully repaired, and nonunion was evident. The bony density for this group was 0.23 ± 0.07 g/cm(2), which was significantly lower than that of the control group (P < 0.05). The β-TCP group was largely held by fibrous tissues. CONCLUSIONS Engineered bone from induced BMSCs and 3D biodegradable β-TCP can efficiently repair critical-sized segmental orbital defects in dogs.