The peripheral vestibular organs are sensors for linear acceleration (gravity and head tilt) and rotation,and turn them into nerve signals that travel to the central nervous system to regulate physiological functions, which play an important role in regulating body stability, ocular movement, autonomic nerve activity, arterial pressure, body temperature, as well as muscle and bone metabolism. The effect of gravity on these functions can be attributed to high plasticity of the vestibular system. In this review, changes in vestibular-related physiological functions induced by the hypergravity and microgravity were introduced, including arterial pressure,muscle and bone metabolism, feeding behavior and body temperature, with the aim to better understand the physiological function of vestibular in adaption to special gravity environment.

Objective To establish the model of goat intervertebral disc degeneration （IDD） induced by controllable axial compressive stress and evaluate its imaging and pathological characteristics. Methods Twenty goats were randomly divided into 4 groups (control group, 4-week pressure group, 8-week pressure group, 12-week pressure group, n=5, 40 N pressure). Disc height index (DHI) was used to evaluate the change of intervertebral disc height by X-ray, Pfirrmann classification method was used to observe the degree of intervertebral disc degeneration by magnetic resonance imaging (MRI), and histopathological observation and evaluation for intervertebral disc were conducted by HE staining and immunohistochemistry. Results DHI in control group showed no significant changes with the extension of pressure time, while DHI in the experimental group gradually decreased. There was no significant change in Pfirrmann classification in control group. In experimental group, with the extension of time, the higher the degeneration aggravated with the Pfirrmann classification increasing. In experimental group, HE staining showed that the disc nucleus pulposus decreased in volume and nucleus pulposus cells, which were gradually replaced by fibrous tissues. Immunohistochemical staining showed that type I collagen in the nucleus pulposus gradually increased, type Ⅱ collagen gradually decreased, and intervertebral disc degeneration occurred. Conclusions A certain axial compressive stress can lead to degeneration of goat lumbar intervertebral disc, and the degree of degeneration is gradually increased with the extension of time.

Prosthetic loosening and periprosthetic inflammation, as serious complications after joint replacement surgery, often require the secondary surgery for repair, which is easy to adversely affect the physical/mental health and economic status of patients.Studies have shown that the functional phenotype expressed by macrophages by different stimuli, namely macrophage polarization state, prolonged M1 polarization can lead to the continuation of long-term inflammation, while timely and effective M2 macrophage phenotype will lead to enhanced osteogenesis and tissue remodeling cytokine secretion and subsequent osseointegration, which play a crucial role in the development and outcome of prosthetic loosening and periprosthetic inflammation.The local micro-environment of extracellular matrix (ECM) is an important factor in the activation, migration, proliferation and fusion of macrophages. Researchers have deeply understood it mainly through the crosstalk between surface properties of biomaterials and macrophages. As an effector cell, macro-phages can perform complex spatiotemporal cellular functional responses by sensing the physical and chemical environment (surface topography, wettability, chemical composition, biological proteins) represented by surface properties of biomaterials.This paper summarizes the recent findings on macrophage polarization and material surface properties.

Objective To investigate the effect of microgravity on MC3T3-E1 osteoblast differentiation. Methods The differential miRNA and mRNA expression profiling of MC3T3-E1 cells during exposure to microgravity were established by RNA transcriptome sequencing technology (RNA-seq). The RNA sequencing results were validated using quantitative real-time polymerase chain reaction (q-PCR). Bioinformatic analyses were applied for further study of these differentially expressed miRNAs and mRNAs. Results Compared with control (CON) group, A total of 1 912 coding transcripts and 160 miRNAs were detected along with osteogenic differentiation in simulated microgravity (SMG) group. Bioinformatic analysis revealed 10 core regulatory genes including 7 mRNAs and 3 miRNAs. Based on the analysis and verification, one miRNA, miR-9_6666-5p, was identified, which might play an important role in osteogenic differentiation process under microgravity. Conclusions The process of osteoblast differentiation was repressed under microgravity which might be related to the changed expression profile of miRNA/mRNA. The research findings can contribute to the better understanding of the molecular mechanisms of mRNA and miRNAs in osteogenic differentiation and bone formation under the microgravity condition.

Objective To design a novel strain loading device for studying the mechanical biology of adherent cells. Methods Based on the technology of substrate deformation loading, the device adopted controllable stepper to cause deformation of the silastic chamber, so as to realize cell loading with multiple units and large strain. The device was developed to test its loading functions. The three-dimensional (3D) models of the silastic chamber were established to simulate the loaded chamber by the finite element technology, and uniformity of the strain field was analyzed. The device applied 5% strain to bone marrow stromal cells (BMSCs) with 0.5 Hz stretch frequency at 2 hours per day for 5 days, and an inverted phase contrast microscope was used to observe the morphology of BMSCs. Results The developed strain loading device for adherent cells in vitro could provide mechanical unidirectional strain up to 50% with three groups of cell loading substrates; within the 10% stain range, the area of uniform strain filed on the silastic chamber remained above 50%, which ensured that the cells were loaded evenly; the morphology of BMSCs was obviously altered, and the direction of arrangement tended to be perpendicular to the loading direction of principal strain. Conclusions The device shows the advantages of reliable operation, wide strain range, adjustable frequency and convenient operation. It can be used to load multiple cell culture substrates at the same time, which provides convenient conditions for the study of cell mechanobiology.

With the development of the 3rd-generation high-throughput sequencing technology and tissue engineering, recent studies show that many long-chain non-coding RNAs (LncRNAs) have played an important role in osteogenic differentiation of mesenchymal stem cells (MSCs). LncRNAs, which are involved in the regulation of mechanical regulation, further regulate bone-related cell functions and play a regulatory role at multiple levels, including transcription, post-transcriptional and epigenetic. LncRNAs may be involved in the osteogenic differentiation and bone remodeling of MSCs, the regulation of bone-related cell functions as a mechanical response molecule, as well as the pathological process of skeletal diseases.

Objective:To establish a hypergravity loading model with a high-acceleration centrifugal loading device and to investigate the effects of different hypergravity loading and icariin on osteoblast adhesion and cytoskeleton.Methods:MC3T3-E1 cells were seeded in the dishes of cell culture at a density of 2×10 5/cm 2. And the experiment was divided into 6 groups: control group (without icariin and loading); simple administration group (only icariin); 10 G loading group (only loading); 10 G administration group (with icariin and loading); 40 G loading group (only loading); 40 G administration group (with icariin and loading). The experimental loading group was loaded with MC3T3-E1 cells using a high-acceleration centrifugal loader. And continuous loading for 3 d, 30 min per d. The control group and the simple administration group were exposed to normal gravity, and the remaining conditions were not different from the experimental group. Icariin was used at a concentration of 10 -7 mol/L in all administration groups, and the experiments were carried out according to the method of preventive administration. At the same time, the related molecular biological techniques such as alizarin red staining, alkaline phosphatase (ALP) activity measurement, CCK-8 cell proliferation experiment, cytoskeleton phalloidin staining, qPCR and Western Blot were used to detect the effects of icariin on osteoblasts adhesion protein integrin α5 and integrin β1 and cytoskeleton protein F-actin under hypergravity extreme mechanical environment. Results:All models were successfully prepared. The alizarin red staining: The icariin could significantly promote the formation of osteoblastic calcified nodules. And the 10 G loading could also promote the mineralization of osteoblasts and increase the number of mineralized nodules, while the mineralization and the number of mineralized nodules of osteoblasts are significantly reduced in 40 G loading. ALP activity test: The OD values of simple administration group, 10 G loading group and 40 G loading group were 0.246, 0.331 and 0.163, respectively. Compared with 0.207 in the control group, the differences were statistically significant ( P<0.05). The 10 G administration group and the 40 G administration group were 0.373 and 0.180, and the differences were statistically significant ( P<0.05). The results of CCK-8 proliferation experiments: The OD value of simple administration group were 0.650, which was statistically significant compared with 0.551 of control group ( P=0.031). The 10 G loading group and 40 G loading group were 1.193 and 0.245, and their differences with the control group were both statistically significant ( P<0.05). The OD value of 10G administration group and the 40 G administration group were 1.300 and 0.310, which were significantly different from the respective loading groups ( P<0.05). Phalloidin staining: 10 G loading significantly increased the number of cells, but the changes in cells morphology and skeleton were not obvious. 40 G loading significantly inhibited the increase of the number of cells, meanwhile, made the pseudopods of cells more shorter and even disappeared. 40 G loading made the seriously damage of the cytoskeleton and even cause the cells to death. Icariin had no effect on the cells morphology, but it did has a certain repair effect after the cells loading. The results of qPCR and Western Blot experiments all confirmed that the expressions of integrin α5, integrin β1 and F-actin were up-regulated after icariin treatment. 10 G loading could promote the expression of integrin α5, integrin β1 and F-actin, and 40 G loading significantly inhibited the expression of the mRNA and proteins. Conclusion:Both 10 G condition and icariin can promote the development, cell adhesion and the cytoskeleton's stability of osteoblasts, while 40 G has a significant inhibitory effect.

In this study, we aim to investigat the effect of microgravity on osteoblast differentiation in osteoblast-like cells (MC3T3-E1). In addition, we explored the response mechanism of nuclear factor-kappa B (NF-κB) signaling pathway to "zero- " in MC3T3-E1 cells under the simulated microgravity conditions. MC3T3-E1 were cultured in conventional (CON) and simulated microgravity (SMG), respectively. Then, the expression of the related osteoblastic genes and the specific molecules in NF-κB signaling pathway were measured. The results showed that the mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin (OCN) and type Ⅰ collagen (CoL-Ⅰ) were dramatically decreased under the simulated microgravity. Meanwhile, the NF-κB inhibitor α (IκB-α) protein level was decreased and the expressions of phosphorylation of IκB-α (p-IκB-α), p65 and phosphorylation of p65 (p-p65) were significantly up-regulated in SMG group. In addition, the IL-6 content in SMG group was increased compared to CON. These results indicated that simulated microgravity could activate the NF-κB pathway to regulate MC3T3-E1 cells differentiation.
3T3 Cells ; Animals ; Cell Differentiation ; Mice ; NF-kappa B ; physiology ; Osteoblasts ; Signal Transduction ; Weightlessness Simulation

BACKGROUND: The silk fibroin/type II collagen composite scaffold has been prepared by low-temperature bio-3D printing technology in the previous study and the scaffold has good mechanical properties. Studies have shown that mechanical stimulation is beneficial to bone remodeling, and gradient loading strain is beneficial to the activation of osteoblasts and osteoclasts. OBJECTIVE: To co-culture silk fibroin/type II collagen composite scaffolds with chondrocytes under compression loading, to observe the proliferation of cells, and to observe the preliminary repair effect of silk fibroin/type II collagen composite scaffold on cartilage defects. METHODS: The silk fibroin/type II collagen composite scaffold was prepared by low-temperature 3D printing to detect the porosity of the scaffold. The passage 3 mouse chondrocytes ADTC-5 were inoculated on the silk fibroin/type II collagen composite scaffold and cultured under static culture and mechanical load respectively. (1) Static culture: blank scaffold was set as control, and cell proliferation was detected by MTT assay at 1, 3, 5, 7, 10, 14 days of inoculation. (2) Culture under mechanical load: blank scaffold was set as control. At 1 day after inoculation, 0％, 1％, 5％, 10％, 15％, 20％ compressive strains were applied to the cell-scaffold complex, and continued to load for 3 days. Cell proliferation was detected by MTT assay, and the distribution, adhesion and morphology of the cells on the scaffold were observed by scanning electron microscopy and hematoxylin-eosin staining. A cartilage defect of 3.5 mm in diameter was made in the bilateral knee joint of New Zealand rabbits. The silk fibroin/type II collagen composite scaffold was implanted onto the left side, and no material was implanted onto the right side. The repair site was observed at 8 weeks after surgery. RESULTS AND CONCLUSION: (1) The porosity of the scaffold was (89.3±3.26)％, which was conducive to cell attachment. (2) After 5 days of static culture, the chondrocytes proliferated well on the surface of the composite scaffold. Under 0％, 1％, 5％, 10％, 15％, 20％ compressive strains, the cell proliferation on the scaffold first increased and then decreased, wherein the cell proliferation was highest under 10％ compressive strain, and lowest under 20％ compressive strain. (4) Under the scanning electron microscopy, the chondrocytes in the 0％ load group were distributed in the surface of the scaffold with irregularities, the cell morphology was obvious, and the cell protrusions were fully extended. There were few or no chondrocytes on the contact surface of the 10％ load group, and more cells distributed on the lateral and internal surfaces of the first layer, but the cell morphology was flat with obvious protrusions. (5) Hematoxylin-eosin staining showed that the chondrocytes in the 0％ load group were concentrated on the surface of the scaffold, and there were almost no cells in the pores, while the chondrocytes in the 10％ load group were distributed in the scaffold pores. (6) There was still a circular defect model with no scaffold implantation, and no obvious repair appeared; similar hyaline cartilage appeared in the defect after scaffold implantation, but there was no adhesion to the surrounding defected cartilage, and the new hyaline cartilage was independent. Overall, the adsorption, proliferation and growth of chondrocytes on the silk fibroin-type II collagen scaffolds is better when the compressive strain is 10％, and the composite scaffold can be used as a repair material for cartilage defects.

Objective To construct a two-dimensional (2D) composite membrane and a three-dimensional (3D) biomimetic scaffold by silk fibroin (SF), type I collagen (Col-I) and hydroxyapatite (HA) blends in vitro, so as to study its physicochemical properties, as well as biocompatibility and explore the feasibility of its application in tissue engineering scaffold materials. Methods 2D composite membranes and 3D scaffolds were prepared by blending SF/Col-I/HA at the bottom of cell culture chamber and low temperature 3D printing combined with vacuum freeze drying. The biocompatibility was evaluated by mechanical property testing, scanning electron microscope and Micro-CT to examine the physicochemical properties of the material, and cell proliferation was detected to evaluate its biocompatibility. Results Stable 2D composite membrane and 3D porous structural scaffolds were obtained by blending and low temperature 3D printing. The mechanical properties were consistent. The pore size, water absorption, porosity and elastic modulus were all in accordance with the requirements of constructing tissue engineering bone. The scaffold was a grid-like white cube with good internal pore connectivity; HA was evenly distributed in the composite membrane, and the cells were attached to the composite membrane in a flat shape; the cells were distributed around pore walls of the scaffold. The shape of the shuttle was fusiform, and the growth and proliferation were good. Conclusions The composite membrane and 3D scaffold prepared by SF/Col-I/HA blending system had better pore connectivity and pore structure, which was beneficial to cell and tissue growth and nutrient transport. Its physicochemical properties and biocompatibility could meet the requirements of bone tissue engineering biomaterials.