Periodic static compression of micro-strain pattern regulates endochondral bone formation.

Introduction: Developmental engineering based on endochondral ossification has been proposed as a potential strategy for repairing of critical bone defects. Bone development is driven by growth plate-mediated endochondral ossification. Under physiological conditions, growth plate chondrocytes undergo compressive forces characterized by micro-mechanics, but the regulatory effect of micro-mechanical loading on endochondral bone formation has not been investigated. Methods: In this study, a periodic static compression (PSC) model characterized by micro-strain (with 0.5% strain) was designed to clarify the effects of biochemical/mechanical cues on endochondral bone formation. Hydrogel scaffolds loaded with bone marrow mesenchymal stem cells (BMSCs) were incubated in proliferation medium or chondrogenic medium, and PSC was performed continuously for 14 or 28 days. Subsequently, the scaffold pretreated for 28 days was implanted into rat femoral muscle pouches and femoral condylar defect sites. The chondrogenesis and bone defect repair were evaluated 4 or 10 weeks post-operation. Results: The results showed that PSC stimulation for 14 days significantly increased the number of COL II positive cells in proliferation medium. However, the chondrogenic efficiency of BMSCs was significantly improved in chondrogenic medium, with or without PSC application. The induced chondrocytes (ichondrocytes) spontaneously underwent hypertrophy and maturation, but long-term mechanical stimulation (loading for 28 days) significantly inhibited hypertrophy and mineralization in ichondrocytes. In the heterotopic ossification model, no chondrocytes were found and no significant difference in terms of mineral deposition in each group; However, 4 weeks after implantation into the femoral defect site, all scaffolds that were subjected to biochemical/mechanical cues, either solely or synergistically, showed typical chondrocytes and endochondral bone formation. In addition, simultaneous biochemical induction/mechanical loading significantly accelerated the bone regeneration. Discussion: Our findings suggest that microstrain mechanics, biochemical cues, and in vivo microenvironment synergistically regulate the differentiation fate of BMSCs. Meanwhile, this study shows the potential of micro-strain mechanics in the treatment of critical bone defects.

Nidogen1-enriched extracellular vesicles accelerate angiogenesis and bone regeneration by targeting Myosin-10 to regulate endothelial cell adhesion.

The technique bottleneck of repairing large bone defects with tissue engineered bone is the vascularization of tissue engineered grafts. Although some studies have shown that extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BMSCs) promote bone healing and repair by accelerating angiogenesis, the effector molecules and the mechanism remain unclear, which fail to provide ideas for the future research and development of cell-free interventions. Here, we found that Nidogen1-enriched EV (EV-NID1) derived from BMSCs interferes with the formation and assembly of focal adhesions (FAs) by targeting myosin-10, thereby reducing the adhesion strength of rat arterial endothelial cells (RAECs) to the extracellular matrix (ECM), and enhancing the migration and angiogenesis potential of RAECs. Moreover, by delivery with composite hydrogel, EV-NID1 is demonstrated to promote angiogenesis and bone regeneration in rat femoral defects. This study identifies the intracellular binding target of EV-NID1 and further elucidates a novel approach and mechanism, thereby providing a cell-free construction strategy with precise targets for the development of vascularized tissue engineering products.

[Cytoskeleton remodeling of rat bone marrow mesenchymal stem cells induced by fibroblast growth factor 2].

Objective To investigate the effects of fibroblast growth factor 2 (FGF-2) on the cytoskeleton and morphology of rat bone marrow mesenchymal stem cells (BMSCs). Methods Morphological and cytoskeleton changes of BMSCs were observed by scanning electron microscopy and rhodamine-phalloidin staining in TranswellTM co-culture system of rat vascular endothelial cells (RAECs) and BMSCs. The content of FGF-2 in cell supernatants were detected by ELISA, and the mRNA expression of FGF-2 in both conventional and co-cultured cells were evaluated by real-time quantitative PCR. NVP-BGJ398, an inhibitor of FGF-2 receptor was added into the co-culture system to block FGF-2 signal and its effect on BMSCs skeleton was observed. Recombinant FGF-2 was supplemented into the conventional medium of BMSCs to further verify the effect of exogenous FGF-2. Results After co-cultured with RAECs, BMSCs gradually stretched, contracted and formed a large number of filopodia. The content of FGF-2 increased in the co-culture system and was mainly secreted by RAECs. Cytoskeleton remodeling of BMSCs was significantly blocked by the inhibitor of FGF-2 receptor and the cells were mostly short spindle-shaped and arranged in a spiral pattern. Exogenous FGF-2 promoted the contraction and edge stretching of BMSCs, forming filopodia with staggered distribution. Conclusion FGF-2 secreted by RAECs induces cytoskeletal remodeling of BMSCs.

[Effects of Different Preservation Methods on Mechanical Properties of Mouse Femur].

In order to establish the best procedure to store the femur samples from the biomechanical viewpoint,we compared the effects of different storage methods on the mechanical properties of mouse femurs.We obtained femurs surgically from twenty C57BL/6Jfemale mice,12 weeks old,and randomly divided them into 5groups,i.e.fresh control group,4% paraformaldehyde fixation group,4℃storage group,-20℃storage group and-80℃storage group,respectively,with five mice in each group.For the three low-temperature storage groups,each group was stored for 1week,2months,6months at their respective temperatures.After rewarming,three-point bending test was performed to test the load and deflection changes.The results showed that both the elastic modulus and deflection decreased significantly in the 4% paraformaldehyde group.The maximum load and elastic modulus of the samples in the 4 ℃ group after one week storage was significantly reduced;The mechanical properties were close to the fresh control group in the-20℃ group stored for 2months but the maximum load was also reduced after 6months.However,mechanical properties,such as elastic load,maximum load and elastic modulus,were not changed obviously in the-80 ℃ storage group.Accordingly,-80 ℃ cryopreservation had little influence on the mechanical properties of bone tissues,which proved that the temperature-80 ℃is a suitable one for long-term preservation.

[Effect of Sox2 on proliferation of cervical squamous cancer cell line SiHa].

OBJECTIVE: To investigate the effect and its mechanism of stem cell related transcription factor Sox2 on the proliferation of cervical squamous carcinoma cell line SiHa. METHODS: Plasmid pIRES-EGFP-Sox2 or empty plasmid (pIRES-EGFP-empty) was stably transfected into SiHa cells. The expression of Sox2 was detected by both RT-PCT and Western blot. The effects of Sox2 on cellular proliferation and cell cycle were studied by MTT assay and flow cytometry (FCM) respectively. The expression of cell cycle related protein CyclinD1 was detected by Western blot. RESULTS: Compared to SiHa-EGFP cells, the expression of Sox2 was obviously up-regulated in SiHaSox2 cells (P < 0.01). MTT result showed that SiHa-Sox2 cells grew faster than the control cells. The over expression of Sox2 increased the proportion of transfected cells in phase S. The increased expression of CyclinD1 was further detected after the successful expression of Sox2 (P < 0.05). CONCLUSION: Sox2 could enhance the proliferation of cervical squamous cancer cells in the manner of up-regulating CyclinD1 expression.