Sensory nerves directly promote osteoclastogenesis by secreting peptidyl-prolyl cis-trans isomerase D (Cyp40).

Given afferent functions, sensory nerves have recently been found to exert efferent effects and directly alter organ physiology. Additionally, several studies have highlighted the indirect but crucial role of sensory nerves in the regulation of the physiological function of osteoclasts. Nonetheless, evidence regarding the direct sensory nerve efferent influence on osteoclasts is lacking. In the current study, we found that high levels of efferent signals were transported directly from the sensory nerves into osteoclasts. Furthermore, sensory hypersensitivity significantly increased osteoclastic bone resorption, and sensory neurons (SNs) directly promoted osteoclastogenesis in an in vitro coculture system. Moreover, we screened a novel neuropeptide, Cyp40, using an isobaric tag for relative and absolute quantitation (iTRAQ). We observed that Cyp40 is the efferent signal from sensory nerves, and it plays a critical role in osteoclastogenesis via the aryl hydrocarbon receptor (AhR)-Ras/Raf-p-Erk-NFATc1 pathway. These findings revealed a novel mechanism regarding the influence of sensory nerves on bone regulation, i.e., a direct promoting effect on osteoclastogenesis by the secretion of Cyp40. Therefore, inhibiting Cyp40 could serve as a strategy to improve bone quality in osteoporosis and promote bone repair after bone injury.

The COVID-19 vaccination decision-making preferences of elderly people: a discrete choice experiment.

COVID-19 is a continuing threat to global public health security. For elderly people, timely and effective vaccination reduces infection rates in this group and safeguards their health. This paper adopted an offline Discrete Choice Experiment (DCE) to research the preference for COVID-19 vaccination amongst Chinese adults aged 50 years and above. Through multinomial logistic regression analysis, our DCE leverages five attributes-the risk of adverse reactions, protective duration, injection doses, injection period, and effectiveness-each of which is split into three to four levels. The risk of adverse reaction and the protective duration were demonstrated to be determinants of vaccination preference. Moreover, it was found that socio demographic factors like region, self-health assessment and the number of vaccinated household members can strengthen or weaken the effects of vaccine attributes. In conclusion, the preferences of the elderly population should be considered when developing COVID-19 vaccination programs for this population in China. Accordingly, the results may provide useful information for policymakers to develop tailored, effectively vaccination strategies.

Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System.

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

Vascularization converts the lineage fate of bone mesenchymal stem cells to endothelial cells in tissue-engineered bone grafts by modulating FGF2-RhoA/ROCK signaling.

The prevascularization of tissue-engineered bone grafts (TEBGs) has been shown to accelerate capillary vessel ingrowth in bone defect remodeling and to enhance new bone formation. However, the exact mechanisms behind this positive effect remain unknown. Here, we report that basic fibroblast growth factor (FGF2)-Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling functions as a molecular switch to regulate the lineage fate of bone mesenchymal stem cells (BMSCs) and that prevascularization promotes the cell fate switch, which contributes to increased bone regeneration with the use of prevascularized TEBGs compared with control TEBGs. Prevascularized TEBGs enhanced the in vivo endothelial differentiation of BMSCs by inhibiting RhoA/ROCK signaling. In vitro data more clearly showed that BMSCs differentiated into von Willebrand factor (vWF)-positive endothelial cells, and FGF2-induced inhibition of RhoA/ROCK signaling played a key role. Our novel findings uncovered a new mechanism that stimulates the increased vascularization of engineered bone and enhanced regeneration by promoting the endothelial differentiation of BMSCs implanted in TEBGs. These results offer a new molecular target to regulate TEBG-induced bone regeneration.

Prevascularization promotes endogenous cell-mediated angiogenesis by upregulating the expression of fibrinogen and connective tissue growth factor in tissue-engineered bone grafts.

BACKGROUND: Vascularization is one of the most important processes in tissue-engineered bone graft (TEBG)-mediated regeneration of large segmental bone defects. We previously showed that prevascularization of TEBGs promoted capillary vessel formation within the defected site and accelerated new bone formation. However, the precise mechanisms and contribution of endogenous cells were not explored. METHODS: We established a large defect (5 mm) model in the femur of EGFP+ transgenic rats and implanted a ß-tricalcium phosphate (ß-TCP) scaffold seeded with exogenous EGFP- cells; the femoral vascular bundle was inserted into the scaffold before implantation in the prevascularized TEBG group. Histopathology and scanning electron microscopy were performed and connective tissue growth factor (CTGF) and fibrin expression, exogenous cell survival, endogenous cell migration and behavior, and collagen type I and III deposition were assessed at 1 and 4 weeks post implantation. RESULTS: We found that the fibrinogen content can be increased at the early stage of vascular bundle transplantation, forming a fibrin reticulate structure and tubular connections between pores of ß-TCP material, which provides a support for cell attachment and migration. Meanwhile, CTGF expression is increased, and more endogenous cells can be recruited and promote collagen synthesis and angiogenesis. By 4 weeks post implantation, the tubular connections transformed into von Willebrand factor-positive capillary-like structures with deposition of type III collagen, and accelerated angiogenesis of endogenous cells. CONCLUSIONS: These findings demonstrate that prevascularization promotes the recruitment of endogenous cells and collagen deposition by upregulating fibrinogen and CTGF, directly resulting in new blood vessel formation. In addition, this molecular mechanism can be used to establish fast-acting angiogenesis materials in future clinical applications.

Nano-biphasic calcium phosphate/polyvinyl alcohol composites with enhanced bioactivity for bone repair via low-temperature three-dimensional printing and loading with platelet-rich fibrin.

BACKGROUND AND AIM: As a newly emerging three-dimensional (3D) printing technology, low-temperature robocasting can be used to fabricate geometrically complex ceramic scaffolds at low temperatures. Here, we aimed to fabricate 3D printed ceramic scaffolds composed of nano-biphasic calcium phosphate (BCP), polyvinyl alcohol (PVA), and platelet-rich fibrin (PRF) at a low temperature without the addition of toxic chemicals. METHODS: Corresponding nonprinted scaffolds were prepared using a freeze-drying method. Compared with the nonprinted scaffolds, the printed scaffolds had specific shapes and well-connected internal structures. RESULTS: The incorporation of PRF enabled both the sustained release of bioactive factors from the scaffolds and improved biocompatibility and biological activity toward bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Additionally, the printed BCP/PVA/PRF scaffolds promoted significantly better BMSC adhesion, proliferation, and osteogenic differentiation in vitro than the printed BCP/PVA scaffolds. In vivo, the printed BCP/PVA/PRF scaffolds induced a greater extent of appropriate bone formation than the printed BCP/PVA scaffolds and nonprinted scaffolds in a critical-size segmental bone defect model in rabbits. CONCLUSION: These experiments indicate that low-temperature robocasting could potentially be used to fabricate 3D printed BCP/PVA/PRF scaffolds with desired shapes and internal structures and incorporated bioactive factors to enhance the repair of segmental bone defects.

Novel standardized massive bone defect model in rats employing an internal eight-hole stainless steel plate for bone tissue engineering.

Massive bone defects are a challenge in orthopaedic research. Defective regeneration leads to bone atrophy, non-union of bone, and physical morbidity. Large animals are important models, however, production costs are high, nursing is complex, and evaluation methods are limited. A suitable laboratory animal model is required to explore the underlying molecular mechanism and cellular process of bone tissue engineering. We designed a stainless steel plate with 8 holes; the middle 2 holes were used as a guide to create a standardized critical size defect in the femur of anaesthetized rats. The plate was fixed to the bone using 6 screws, serving as an inner fixed bracket to secure a tricalcium phosphate implant seeded with green fluorescent protein-positive rat bone marrow mesenchymal stem cells within the defect. In some animals, we also grafted a vessel bundle into the lateral side of the implant, to promote vascularized bone tissue engineering. X-ray, microcomputed tomography, and histological analyses demonstrated the stainless steel plate resulted in a stable large segmental defect model in the rat femur. Vascularization significantly increased bone formation and implant degradation. Moreover, survival and expansion of green fluorescent protein-positive seeded cells could be clearly monitored in vivo at 1, 4, and 8 weeks postoperation via fluorescent microscopy. This standardized large segmental defect model in a small animal may help to advance the study of bone tissue engineering. Furthermore, availability of antibodies and genetically modified rats could help to dissect the precise cellular and molecular mechanisms of bone repair.

CD31hiEmcnhi Vessels Support New Trabecular Bone Formation at the Frontier Growth Area in the Bone Defect Repair Process.

CD31hiEmcnhi vessels were a subtype of vessels in the murine skeletal system, with high levels of platelet and endothelial cell adhesion molecule-1 (PECAM-1/CD31) and endomucin (Emcn). They were reported coupling angiogenesis and osteogenesis during bone development. We investigated the distribution of these vessels in rat tibiae and their temporal and spatial distribution during the bone defect repair process to improve our understanding of the importance of these vessels. We confirmed that CD31hiEmcnhi vessels were specially distributed around the trabecular bones near metaphysis and endosteum in rat tibiae. At 3 days post bone injury, CD31hiEmcnhi vessels proliferated and were extensively distributed across the entire repair area. At 7 and 14 days post-injury, these vessels decreased but were specially distributed around the growing trabecular bones near the frontier growth area, suggesting that these vessels support new bone formation. The distribution of CD31hiEmcnhi vessels and the transcriptions of Hif-1α and VEGFA, as well as BMP2 and Osterix decreased at 7 and 14 days post-injury under osteoporotic conditions, in combination with insufficient osteogenesis. Our research is of great significance to help understand the important role of CD31hiEmcnhi vessels in supporting new trabecular bones formation during bone defect repair process.

[Effect of micro-ecological environment on incidence of allergic rhinitis on mice].

OBJECTIVE: This study was designed to find out the impact of micro-ecological environment on the incidence of allergic rhinitis after developing a model of allergic rhinitis on mice. METHOD: Sixty mice were randomly divided into GF group (n=30) and SPF group (n=30). Mice of GF group were fed in the germ-free environment and mice of SPF group were fed in the specific pathogen-free environment. Then each group were randomly divided into model group (20 mice) and control group (10 mice). Establish allergic rhinitis model in the mice of model group using ovalbumin (OVA) at the age of 6 weeks, observe and score the corresponding symptoms and signs that could been induced. Stain with hematoxylin eosin (HE) staining method for nasal mucosa to observe the morphological changes. Using enzyme linked immunosorbent assay to detect the concentration of IgE, IFN-γ and IL-4 in the peripheral blood serum. RESULT: The chi square test showed that the incidence of allergic rhinithis in the mice of GF group was significantly higher than that in the SPF group (P< 0. 05). HE staining showed that the nasal mucosas of allergic rhinitis positive reaction mice were highly congestive and edematous and had a large number of inflammatory cell infiltration, while there was no abnormal morphology of nasal mucosas in mice with no allergic rhinitis reaction. EOS counting displayed that the number of eosinophilic cells in nasal mucosa of positive allergic rhinitis reaction mice was increased significantly. The concentration of IgE and IL-4 in the serum of positive allergic rhinitis reaction mice was highly increased (P <0. 05), and IFN-γ was significantly decreased (P< 0.05). CONCLUSION: The difference of micro-ecological environment may play a key role in the occurrence of allergic rhinitis in mice.

The human leukocyte antigen G promotes trophoblast fusion and ß-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines.

The human leukocyte antigen G (HLA-G) is expressed on the fetal-maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell-cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (ß-hCG) were elevated. HLA-G up-regulates ß-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in ß-hCG compared with control cells. The defect in ß-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating ß-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

[One-stage anteroposterior hemi-vertebra resection and segmental internal fixation for young children with congenital scoliosis].

OBJECTIVE: to discuss the feasibility and efficacy of one-stage anteroposterior hemi-vertebra resection and segmental internal fixation for young children with congenital scoliosis. METHODS: thirty-five patients undergoing one-stage anteroposterior hemi-vertebra resection and segmental internal fixation were retrospectively studied. The mean followed-up period was 5.3 years (range: 1.2 - 8.7). The Cobb's angle of scoliosis at pre and post-operation was compared. RESULTS: all children's parents were satisfied with the outcome. The Cobb's angle of scoliosis was corrected from (42.5 ± 6.7)° to (16.2 ± 3.2)° at post-operation. The coronal correction rate was 64.7%. The angle of kyphosis improved from preoperative (33.5 ± 5.2)° to postoperative (13.3 ± 5.6)° in 14 cases. Operative duration was 210 - 280 minutes with an average of 240 minutes. The intra-operative blood loss was 80 - 200 ml with an average of 120 ml. There was no significant correction loss at follow-up. No neurological complication, infection or pedicular fracture was reported. CONCLUSION: the procedure of one-stage anteroposterior hemi-vertebra resection and segmental internal fixation is a safe and effective treatment for scoliosis by congenital hemi-vertebra in young children. A satisfactory correction may be achieved with a short fusion segment.