Both fluid shear stress and matrix stiffness are implicated in bone metabolism and functional adaptation, but the synergistic action of these mechanical cues on the biological behaviors of mesenchymal stem cells (MSCs) is still not well-known. In the present work, a homemade oscillatory flow device was applied to investigate the effects of matrix stiffness on MSCs survival, distribution, and osteogenic differentiation in three-dimensional (3D) conditions. Furthermore, the flow field and cell growth in this bioreactor were theoretically simulated. The results demonstrated that oscillatory shear stress significantly increased the viability and distribution uniformity of MSCs throughout the scaffold after culture for 3 weeks. Compared to static culture, oscillatory shear stress could promote the collagen secretion, mineral deposits, and osteogenic differentiation of MSCs. The findings obtained from this work indicate that the oscillatory perfusion not only provides a higher survival rate and a more uniform distribution of cells but also facilitates osteogenic differentiation of MSCs. Oscillating perfusion bioreactor culture of MSCs in 3D scaffold with optimal matrix stiffness could offer an easy-to-use but efficient bioreactor for bone tissue engineering.Keywords: 3D scaffold; bioreactor; bone tissue engineering; matrix stiffness; mesenchymal stem cells; perfusion culture;

Substrate stiffness and hypoxia are associated with tumor development and progression, respectively. However, the synergy of them on the biological behavior of human breast cancer cell is still largely unknown. This study explored how substrate stiffness regulates the cell phenotype, viability, and epithelial-mesenchymal transition (EMT) of human breast cancer cells MCF-7 under hypoxia (1% O2). TRITC-phalloidin staining showed that MCF-7 cells transformed from round to irregular polygon with stiffness increase either in normoxia or hypoxia. While being accompanied with the upward tendency from a 0.5- to a 20-kPa substrate, the percentage of cell apoptosis was significantly higher in hypoxia than that in normoxia, especially on the 20-kPa substrate. Additionally, it was hypoxia, but not normoxia, that promoted the EMT of MCF-7 by upregulating hypoxia-inducible factor-1α (HIF-1α), vimentin, Snail 1, and matrix metalloproteinase 2 (MMP 2) and 9 (MMP 9), and downregulating E-cadherin simultaneously regardless of the change of substrate stiffness. In summary, this study discovered that hypoxia and stiffer substrate (20 kPa) could synergistically induce phenotype change, apoptosis, and EMT of MCF-7 cells. Results of this study have an important significance on further exploring the synergistic effect of stiffness and hypoxia on the EMT of breast cancer cells and its molecular mechanism.

•Suspension state increases adhesion and reattachment of breast cancer cells.•Suspension state induces lamin A/C accumulation.•Disrupting actin cytoskeleton induces lamin A/C protein accumulation.•Lamin A/C accumulation contributes to reattachment and stiffness of tumor cells.Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis.Download high-res image (263KB)Download full-size image

•MGF pretreatment can weaken the negative effects of severe hypoxia on BMSCs.•MGF E peptide inhibited HIF-1α expression.•Cell proliferation and differentiation were recovered by MGF pretreatment.•MEK-ERK1/2 and PI3K-Akt signaling pathway were involved in MGF regulating of BMSCs.AimsSevere hypoxia always inhibits the cell proliferation, osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and hinders bone defect repair. Herein we explored the effects of mechano-growth factor (MGF) E peptide on the proliferation and osteogenic differentiation of BMSCs under severe hypoxia.Materials and methodsCoCl2 was utilized to simulate severe hypoxia. MTS was used to detect cell viability. Cell proliferation was verified through flow cytometry and EdU assay. Osteogenic differentiation of BMSCs and osteoblast-specific genes were detected through alkaline phosphatase (ALP) and Alizarin Red S staining, and quantitative real-time PCR, respectively. Hypoxia-inducible factor 1α (HIF-1α), p-ERK1/2 and p-Akt expression levels were detected through western blotting and immunofluorescence.Key findingsSevere hypoxia induced HIF-1α accumulation and transferring into the nucleus, and reduced cell proliferation and osteogenic differentiation of BMSCs. The expression levels of osteoblast-specific genes were markedly decreased after differentiation culture for 0, 7 or 14 days. Fortunately, MGF E peptide inhibited HIF-1α expression and transferring into the nucleus. Cell proliferation and osteogenic differentiation of BMSCs could be recovered by MGF E peptide pretreatment. MEK-ERK1/2 and PI3K-Akt signaling pathway were confirmed to be involved in MGF E peptide regulating the abovementioned indexes of BMSCs. What's more, short-time treatment with MGF E peptide alone promoted the osteogenic differentiation of BMSCs as well.SignificanceOur study provides new evidence for the role of MGF E peptide in regulating proliferation and osteogenic differentiation of BMSCs under severe hypoxia, which may potentially have therapeutic implication for bone defect repair.Download high-res image (287KB)Download full-size image

Most tissue engineered bone scaffolds are limited by insufficient vascularization. In this study, a high mobility group box 1 (HMGB1)-immobilized material for in situ tissue engineering was prepared to facilitate bone regeneration. The HMGB1-immobilized scaffold accelerated the adhesion and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. Subcutaneous implantation and rat calvarial defect repair experiments proved that the HMGB1-immobilized scaffold induced vascularization and enhanced expression of osteocalcin in vivo. MSCs were recruited into the scaffold, which might due to the high expression of stromal cell-derived factor-1α. The bone repair efficiency of the HMGB1-immobilized scaffold was significantly superior to and faster than that of the poly-L-lactide/polycaprolactone nanofibrous scaffold. This research demonstrated the feasibility of using only one pro-inflammatory cytokine HMGB1 as a ‘trigger’ signal in bone tissue engineering to simultaneously realize multiple functions, including enhancing vascularization, inducing osteogenesis and recruiting stem cells. Consequently, the bone regeneration process was accelerated. The results are of great significance for preparing a new type of scaffold that can promote bone repair in multiple aspects by mimicking a natural manner.

A growing body of evidence has shown that extracellular matrix (ECM) stiffness can modulate stem cell adhesion, proliferation, migration, differentiation, and signaling. Stem cells can feel and respond sensitively to the mechanical microenvironment of the ECM. However, most studies have focused on classical two-dimensional (2D) or quasi-three-dimensional environments, which cannot represent the real situation in vivo. Furthermore, most of the current methods used to generate different mechanical properties invariably change the fundamental structural properties of the scaffolds (such as morphology, porosity, pore size, and pore interconnectivity). In this study, we have developed novel three-dimensional (3D) scaffolds with different degrees of stiffness but the same 3D microstructure that was maintained by using decellularized cancellous bone. Mixtures of collagen and hydroxyapatite [HA: Ca10(PO4)6(OH)2] with different proportions were coated on decellularized cancellous bone to vary the stiffness (local stiffness, 13.00 ± 5.55 kPa, 13.87 ± 1.51 kPa, and 37.7 ± 19.6 kPa; bulk stiffness, 6.74 ± 1.16 kPa, 8.82 ± 2.12 kPa, and 23.61 ± 8.06 kPa). Microcomputed tomography (μ-CT) assay proved that there was no statistically significant difference in the architecture of the scaffolds before or after coating. Cell viability, osteogenic differentiation, cell recruitment, and angiogenesis were determined to characterize the scaffolds and evaluate their biological responses in vitro and in vivo. The in vitro results indicate that the scaffolds developed in this study could sustain adhesion and growth of rat mesenchymal stem cells (MSCs) and promote their osteogenic differentiation. The in vivo results further demonstrated that these scaffolds could help to recruit MSCs from subcutaneous tissue, induce them to differentiate into osteoblasts, and provide the 3D environment for angiogenesis. These findings showed that the method we developed can build scaffolds with tunable mechanical properties almost without variation in 3D microstructure. These preparations not only can provide a cell-free scaffold with optimal matrix stiffness to enhance osteogenic differentiation, cell recruitment, and angiogenesis in bone tissue engineering but also have significant implications for studies on the effects of matrix stiffness on stem cell differentiation in 3D environments.Keywords: 3D microenvironment; bone tissue engineering; decellularized cancellous bone; matrix stiffness; mesenchymal stem cells; osteogenic differentiation;

To evaluate the effects of the combination of low-intensity pulsed ultrasound (LIPUS) and induced pluripotent stem cells-derived neural crest stem cells (iPSCs-NCSCs) on the regeneration of rat transected sciatic nerve in vivo.Tissue-engineered tubular nerve conduit was fabricated by electrospinning aligned nanofibers in longitudinal direction. This sustained the iPSCs-NCSCs and could be used as a bridge in rat transected sciatic nerve. Treatment with 0.3 W cm−2 LIPUS for 2 weeks and 5 min per day significantly improved the sciatic functional index, static sciatic function index and nerve conduction velocity of rat sciatic nerve. Histological analysis showed that there were more regenerative new blood vessels and new neurofilaments, higher expression level of β-III tubulin (Tuj1) in the experimental group seeded with iPSCs-NCSCs and stimulated with LIPUS. Combination of LIPUS with iPSCs-NCSCs promoted the regeneration and reconstruction of rat transected sciatic nerve and is an efficient and cost-effective method for peripheral nerve regeneration.

Based on our previous study which tested the feasibility of protecting the healthy tissue around the cancerous tissue during cryosurgery by microencapsulated phase change materials (PCMs) with large latent heat and low thermal conductivity, uncertainties and sensitivities for thermal protection efficiency caused by the deviations of the PCM properties’ values, the PCMs concentration and the distance between the PCMs domain and the tumor domain were further investigated in this study. The preliminary results showed that the radius of the micro/nano PCM particle, the upper and lower phase transition temperatures of the PCM and the distance between the PCMs domain and the tumor domain should be accurately measured before performing thermal protection by PCMs during cryosurgery. Less than 20 % deviations of the heat capacities of solid and liquid PCM almost had no obvious influence on the thermal protection efficiency. The results obtained in this study will further help us to optimize the protection protocol by PCMs before performing cryosurgery.

In this study, a new method for thermal protection by microcapsulated phase change micro/nanoparticles during hyperthermia was proposed and a three-dimensional theoretical model was developed to test the feasibility of this method. Effective capacity method was used to get the transient temperature distribution in cancerous tissue embedded with superparamagnetic nanoparticles and healthy tissue embedded with phase change materials (PCMs). The results indicated that embedding micro/nano PCMs can decrease the healthy tissue temperature and has no obvious influence on the temperature in cancerous tissue under the same heating condition, which can help protect the healthy tissue during hyperthermia. The influences of the domain, concentration, latent heat, and the lower and upper phase transition temperatures of the PCM on the thermal protection efficacy were also further analyzed in detail. This study may open a new technical approach for enlarging application of hyperthermia.

Although considerable progresses have been made in cryosurgery to treat tumor, thermal injury to collateral structures is still a known complication of cryosurgery. In this study, a new method was proposed to prevent the healthy tissue around the cancerous tissue from thermal injury by microencapsulated phase change micro/nanoparticles, in which the phase change materials (PCMs) with large latent heat and low thermal conductivity are microencapsulated by liposome and delivered to the healthy tissue by mainline, arterial injection, hypodermic injection or direct injection. The three-dimensional transient temperature field in human body containing one tumor and embedded PCMs in the surrounding healthy tissue was numerically studied. The effects of the PCMs concentration, the phase change temperature, the temperature range near the phase change point, the latent heat and the PCMs distribution (especially the number of sides that PCMs cover, and the distance between the PCMs domain and the cancerous domain) were further discussed. The computational analysis showed that embedding PCMs in the healthy tissue around the cancerous tissue can significantly reduce the cryoinjury to the surrounding healthy tissue. The result also suggested that not embedding the PCMs directly adjacent to the cancerous tissue will help to improve the protection efficacy.Highlights► A new method was proposed to prevent the healthy tissue around the cancerous tissue from cryoinjury. ► Three-dimensional transient temperature field in human body was numerically studied. ► Embedding PCMs in the healthy tissue can reduce the cryoinjury to the surrounding healthy tissue.

Most tissue engineered bone scaffolds are limited by insufficient vascularization. In this study, a high mobility group box 1 (HMGB1)-immobilized material for in situ tissue engineering was prepared to facilitate bone regeneration. The HMGB1-immobilized scaffold accelerated the adhesion and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. Subcutaneous implantation and rat calvarial defect repair experiments proved that the HMGB1-immobilized scaffold induced vascularization and enhanced expression of osteocalcin in vivo. MSCs were recruited into the scaffold, which might due to the high expression of stromal cell-derived factor-1α. The bone repair efficiency of the HMGB1-immobilized scaffold was significantly superior to and faster than that of the poly-L-lactide/polycaprolactone nanofibrous scaffold. This research demonstrated the feasibility of using only one pro-inflammatory cytokine HMGB1 as a ‘trigger’ signal in bone tissue engineering to simultaneously realize multiple functions, including enhancing vascularization, inducing osteogenesis and recruiting stem cells. Consequently, the bone regeneration process was accelerated. The results are of great significance for preparing a new type of scaffold that can promote bone repair in multiple aspects by mimicking a natural manner.