Fisetin orchestrates neuroinflammation resolution and facilitates spinal cord injury recovery through enhanced autophagy in pro-inflammatory glial cells.

BACKGROUND: Neuroinflammation, a critical component of the secondary injury cascade post-spinal cord injury, involves the activation of pro-inflammatory cells and release of inflammatory mediators. Resolution of neuroinflammation is closely linked to cellular autophagy. This study investigates the potential of Fisetin, a natural anti-inflammatory compound, to ameliorate neuroinflammation and confer spinal cord injury protection through the regulation of autophagy in pro-inflammatory cells. METHODS: Utilizing a rat T10 spinal cord injury model with distinct treatment groups (Sham, Fisetin-treated, and Fisetin combined with autophagy inhibitor), alongside in vitro models involving lipopolysaccharide (LPS)-stimulated microglial cell activation and co-culture with neurons, we employed techniques such as transcriptomic sequencing, histological assessments (immunofluorescence staining, etc.), molecular analyses (PCR, WB, ELISA, etc.), and behavioral evaluations to discern differences in neuroinflammation, autophagy, neuronal apoptosis, and neurological function recovery. RESULTS: Fisetin significantly augmented autophagic activity in injured spinal cord tissue, crucially contributing to neurological function recovery in spinal cord-injured rats. Fisetin's autophagy-dependent effects were associated with a reduction in neuronal apoptosis at the injury site. The treatment reduced the population of CD68+ and iNOS+ cells, coupled with decreased pro-inflammatory cytokines IL-6 and TNF-α levels, through autophagy-dependent pathways. Fisetin pre-treatment attenuated LPS-induced pro-inflammatory polarization of microglial cells, with this protective effect partially blocked by autophagy inhibition. Fisetin-induced autophagy in the injured spinal cord and pro-inflammatory microglial cells was associated with significant activation of AMPK and inhibition of mTOR. CONCLUSION: Fisetin orchestrates enhanced autophagy in pro-inflammatory microglial cells through the AMPK-mTOR signaling pathway, thereby mitigating neuroinflammation and reducing the apoptotic effects of neuroinflammation on neurons. This mechanistic insight significantly contributes to the protection and recovery of neurological function following spinal cord injury, underscoring the vital nature of Fisetin as a potential therapeutic agent.

Tofacitinib Promotes Functional Recovery after Spinal Cord Injury by Regulating Microglial Polarization via JAK/STAT Signaling Pathway.

Background: The JAK/STAT signaling pathway is the main inflammatory signal transduction pathway, whether JAK/STAT contributes the pathology of SCI and targeting the pathway will alleviate SCI needs to be addressed. Here, we explored the therapeutic effect of pan-JAK inhibitor tofacitinib (TOF) on secondary injury after SCI and explained the underlying mechanisms. Methods: SCI model in rat was established to evaluate the therapeutic effects of TOF treatment in vivo. Histological and behavioral analyses were performed at different time points after SCI. In vitro, the effects of TOF on pro-inflammatory activation of primary microglia and BV2 cells were analyzed by western blot analysis, fluorescent staining, qPCR and flow cytometry. The neuroprotection of TOF was detected using a co-culture system with primary neurons and microglia. Results: TOF can effectively improve motor dysfunction caused by spinal cord injury in rats. TOF administration in the early stage of inflammation can effectively inhibit neuronal apoptosis and scar tissue formation, and promote the repair of axons and nerve fibers. Further studies have demonstrated that TOF suppresses inflammation caused by spinal cord injury by inhibiting the activation of microglia to pro-inflammatory phenotype in vivo and in vitro. Additionally, an interesting phenomenon is revealed in our results that TOF exhibits superior neuronal protection during inflammation in vitro. Conclusions: Our study showed that TOF could regulate microglial activation via JAK / STAT pathway and promote the recovery of motor function after SCI, which is of great significance for the immunotherapy of SCI.

3D-Printed ß-Tricalcium Phosphate Scaffolds Promote Osteogenic Differentiation of Bone Marrow-Deprived Mesenchymal Stem Cells in an N6-methyladenosine-Dependent Manner.

Bone defect is a serious orthopedic disease which has been studied for a long time. Alternative degradable biomaterials are required for bone repairing and regeneration to address the limitation of autogenous bone. ß-tricalcium phosphate (ß-TCP) is an alternative material with good cytocompatibility and has been used in bone defect treatment. However, whether ß-TCP contributes to osteogenesis of bone marrow stem cells (BMSCs) through N6-methyladenosine (m6A) modification remains unknown. To address this issue, we verified the effects of ß-TCP on osteogenesis of BMSCs. We also studied the expression of m6A-related enzymes in BMSCs after ß-TCP treatment. Furthermore, the m6A level and stability of Runt-related transcription factor 2 (RUNX2) mRNA were investigated after ß-TCP treatment. Finally, rat calvarial defect models were performed to detect expression level of osteogenic factors and m6A-related enzymes after the stimulation of three-dimension (3D)-printed ß-TCP scaffolds. We found that ß-TCP showed good biocompatibility and was osteoinductive. Meanwhile, methyltransferase-like 3 (METTL3) increased, causing the elevation of m6A level of RUNX2, results in stabler RUNX2 mRNA level. At last, based on the animal experiments, we demonstrated that the increase of RUNX2 and METTL3 levels was induced by ß-TCP. These findings suggest that METTL3 increases the m6A level of RUNX2 mRNA after ß-TCP induction, contributing to its stability, and the results in vivo also confirmed the osteogenic and bone-repair properties of ß-TCP.

HIF-1α inhibition attenuates severity of Achilles tendinopathy by blocking NF-κB and MAPK pathways.

Tendinopathy is a common disease influencing life quality and tendon function of patients, especially in the elderly and athletes. Inflammation is an important pathologic process of tendinopathy. Hypoxia inducible factor-1 alpha (HIF-1α) participates actively in inflammatory process. However, little is known about the role of HIF-1α in tendinopathy. To address this issue, we verified the expression level of HIF-1α in tendinopathy in vivo and in vitro. Furthermore, the severity of tendinopathy in vivo and in vitro was assessed after HIF-1α inhibition. At the same time, inflammatory signaling cascades were evaluated. The expression level of HIF-1α increased in tendinopathy in vivo and in vitro. The migration and proliferation of tendon cells (TDCs) were reduced after HIF-1α inhibition. In the meantime, HIF-1α inhibition alleviated the severity of tendinopathy and promoted tendon repairing. We also found that HIF-1α inhibition reduced the phosphorylation levels of p65 in NF-κB signaling pathway and the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and Jun N-terminal kinase (JNK) in MAPK signaling pathway. These findings suggest that HIF-1α increases in tendinopathy in vivo and vitro, and HIF-1α inhibition can suppress the severity of tendinopathy by blocking NF-κB and MAPK signaling pathways.

The effect of enhanced bone marrow in conjunction with 3D-printed PLA-HA in the repair of critical-sized bone defects in a rabbit model.

BACKGROUND: Traditionally, the iliac crest has been the most common harvesting site for autologous bone grafts; however, it has some limitations, including poor bone availability and donor-site morbidity. This study sought to explore the effect of enhanced bone marrow (eBM) in conjunction with three-dimensional (3D)-printed polylactide-hydroxyapatite (PLA-HA) scaffolds in the repair of critical-sized bone defects in a rabbit model. METHODS: First, 3D-printed PLA-HA scaffolds were fabricated and evaluated using micro-computed tomography (µCT) and scanning electron microscopy (SEM). Twenty-seven New Zealand white rabbits were randomly divided into 3 groups (n=9 per group), and the defects were treated using 3D-printed PLA-HA scaffolds (the PLA-HA group) or eBM in conjunction with 3D-printed PLA-HA scaffolds (the PLA-HA/eBM group), or were left untreated (the control group). Radiographic, µCT, and histological analyses were performed to evaluate bone regeneration in the different groups. RESULTS: The 3D-printed PLA-HA scaffolds were cylindrical, and had a mean pore size of 500±47.1 µm and 60%±3.5% porosity. At 4 and 8 weeks, the lane-sandhu X-ray score in the PLA-HA/eBM group was significantly higher than that in the PLA-HA group and the control group (P<0.01). At 8 weeks, the µCT analysis showed that the bone volume (BV) and bone volume/tissue volume (BV/TV) in the PLA-HA/eBM group were significantly higher than those in the PLA-HA group and the control group (P<0.01). Hematoxylin and eosin staining indicated that the new bone area in the PLA-HA/eBM group was significantly higher than that in the PLA-HA group and the control group (P<0.01). CONCLUSIONS: The group that was treated with eBM in conjunction with 3D-printed PLA-HA showed enhanced bone repair compared to the other 2 groups. PLA-HA/eBM scaffolds represent a promising way to treat critical-sized bone defects.

Use of a novel Screen-Enrich-Combine(-biomaterials) Circulating System to fill a 3D-printed open Ti6Al4V frame with mesenchymal stem cells/ß-tricalcium phosphate to repair complex anatomical bone defects in load-bearing areas.

BACKGROUND: Repairing complex anatomical load-bearing bone defects is difficult because it requires the restoration of the load-bearing function, reconstructing the anatomical shape, and repair by regenerated bone. We previously developed a Screen-Enrich-Combine(-biomaterials) Circulating System (SECCS) for rapid intraoperative enrichment of autologous bone marrow mesenchymal stem cells (MSCs) to enhance the osteogenic ability of porous bone substitutes. In this study, we prepared a 3D-printed Ti6A14V macroporous frame matching the defect shape to provide early load-bearing support and evaluated the efficacy of filling the frame with SECCS-processed MSCs/beta tricalcium phosphate (ß-TCP) for long-term bone growth. METHODS: Fifteen 2-year-old goats were involved in this study, and the lateral part of their distal femur was removed by an electric saw and was fitted by a matching electron beam melting technology-prepared (EBM) Ti6Al4V frame. Three types of frames, filled with nothing, pure porous ß-TCP, or SECCS-processed MSCs/ß-TCP, were fixed onto the defect site. Repair efficacy was evaluated by X-ray radiography, computed tomography (CT), histology, and histomorphometry. RESULTS: In the basic regular hexagon printing unit, the combined side width (w) and the inscribed circle diameter (d) determines the printing frame's mechanical strength. The compressive load was significantly higher for w=1.9 mm, d=4.4 mm than for w=1.7 mm, d=4.0 mm or w=2.0 mm, d=5.0 mm (P<0.05). The EBM-prepared Ti6Al4V defect-matched frame was well maintained 9 months after implantation. The MSCs successfully adhered to the wall of the porous ß-TCP in the SECCS-processed group and had spread fully in the test samples. Each goat in the MSCs/ß-TCP-the filled group, had approximately 31,321.7±22,554.7 of MSCs and a larger area of new bone growth inside the frame than the control and blank areas groups. CONCLUSIONS: Filling the 3D-printed Ti6Al4V large-aperture frame with osteogenic materials achieved biological reconstruction over a larger area of regenerated bone to repair complex anatomical weight-bearing bone defects under the condition of early frame-supported load bearing. MSCs/ß-TCP prepared by SECCS can be used as a filling material for this type of bone defect to obtain more efficacious bone repair.

Exposure to high levels of magnesium disrupts bone mineralization in vitro and in vivo.

BACKGROUND: The removal of permanent internal fixation devices by secondary surgery could be avoided if these devices were made of degradable magnesium and magnesium alloys. Before such implants can be used clinically, however, the biological effect of magnesium exposure on surrounding bone must be evaluated. Previous studies have focused on bone formation; few have examined the effects of magnesium on the bone quality that affect many biomechanical properties. METHODS: Using bone quality parameters, we analyzed in vivo changes in bone properties and biomechanics after exposure to locally high levels of magnesium. RESULTS: Local bone mineralization was significantly disrupted following exposure to a porous rod of pure magnesium. Normal crystal formation and crystallinity were inhibited and the mineral-to-matrix ratio decreased. These results were consistent with those of in vitro experiments, in which high levels of magnesium inhibited mineral deposition by mesenchymal stem cells (MSCs) but increased alkaline phosphatase (ALP) expression. The same mineralization inhibition was observed around magnesium implants via micro-computerized tomography (micro-CT) and von Kossa staining. Such reduced bone quality around degrading magnesium rods could negatively impact bone biomechanics. CONCLUSIONS: This study showed that exposure to the local high magnesium levels that arise from rapidly degrading magnesium devices may significantly disrupt bone mineralization and negatively impact bone biomechanics.

LncRNA expression profiles and the negative regulation of lncRNA-NOMMUT037835.2 in osteoclastogenesis.

Bone is a rigid and dynamic organ that continuously undergoes remodeling and repair. The balance between osteoblastic bone formation and osteoclastic bone resorption is essential for normal bone homeostasis. Osteoclasts are giant multinucleated cells derived from the monocyte/macrophage hematopoietic lineage and are regulated by various cytokines. Long non-coding (lnc) RNAs are known to regulate many biological processes in the skeletal system in both normal and diseased states; however, the lncRNA-mediated regulation of osteoclastogenesis has not been extensively studied. Hence, in the present study, we performed microarray analysis of lncRNAs expressed during different stages of osteoclast differentiation and fusion. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed the biological functions of target genes of lncRNAs that were specifically up- or downregulated at the different stages. Microarray and bioinformatic prediction results were used to generate co-expression networks of lncRNAs-mRNAs and lncRNAs-transcription factors. Based on the analysis, we identified one lncRNA, NONMMUT037835.2, which plays an important role during osteoclastogenesis. Upregulation of lncRNA-NONMMUT037835.2 inhibited osteoclastic differentiation, whereas downregulation of lncRNA-NONMMUT037835.2 promoted osteoclast formation and fusion. Our study also indicated that lncRNA-NOMMUT037835.2 might regulated osteoclastogenesis through negatively regulating RANK expression and inhibiting NF-κB/MAPK signaling pathway. Our results lead to a better understanding of the molecular mechanisms and provided a theoretical basis for developing therapeutic agents for diseases related to dysregulation of bone homeostasis.

Comparison and characterization of enriched mesenchymal stem cells obtained by the repeated filtration of autologous bone marrow through porous biomaterials.

BACKGROUND: When bone marrow is repeatedly filtered through porous material, the mesenchymal stem cells (MSCs) in the bone marrow can adhere to the outer and inner walls of the carrier material to become enriched locally, and this is a promising method for MSC enrichment. In this process, the enrichment efficiency of MSCs involved in the regulation of the cell ecology of postfiltration composites containing other bone marrow components is affected by many factors. This study compared the enrichment efficiency and characterized the phenotypes of enriched MSCs obtained by the filtration of autologous bone marrow through different porous bone substitutes. METHODS: Human bone marrow was filtered through representative porous materials, and different factors affecting MSC enrichment efficiency were evaluated. The soluble proteins and MSC phenotypes in the bone marrow before and after filtration were also compared. RESULTS: The enrichment efficiency of the MSCs found in gelatin sponges was 96.1% ± 3.4%, which was higher than that of MSCs found in allogeneic bone (72.5% ± 7.6%) and porous ß-TCP particles (61.4% ± 5.4%). A filtration frequency of 5-6 and a bone marrow/material volume ratio of 2 achieved the best enrichment efficiency for MSCs. A high-throughput antibody microarray indicated that the soluble proteins were mostly filtered out and remained in the flow through fluid, whereas a small number of proteins were abundantly (> 50%) enriched in the biomaterial. In terms of the phenotypic characteristics of the MSCs, including the cell aspect ratio, osteogenetic fate, specific antigens, gene expression profile, cell cycle stage, and apoptosis rate, no significant changes were found before or after filtration. CONCLUSION: When autologous bone marrow is rapidly filtered through porous bone substitutes, the optimal enrichment efficiency of MSCs can be attained by the rational selection of the type of carrier material, the bone marrow/carrier material volume ratio, and the filtration frequency. The enrichment of bone marrow MSCs occurs during filtration, during which the soluble proteins in the bone marrow are also absorbed to a certain extent. This filtration enrichment technique does not affect the phenotype of the MSCs and thus may provide a safe alternative method for MSC enrichment.

Screen-enrich-combine circulating system to prepare MSC/ß-TCP for bone repair in fractures with depressed tibial plateau.

Aim: To evaluate the clinical efficacy of mesenchymal stem cell/ß-tricalcium phosphate composites (MSC/ß-TCP) prepared with a screen-enrich-combine circulating system (SECCS) in patients with depressed tibial plateau fractures. Materials & methods: Bone defects in depressed tibial plateaus were filled with MSC/ß-TCP (n = 16) or with ß-TCP only (n = 23). Enrichment efficiency and effect of enrichment on cell viability were evaluated. Clinical results were assessed by imaging examination and Lysholm score. Results: SECCS effectively integrated MSCs with ß-TCP. At 18 months postimplantation, new bone ratio was significantly higher in patients treated with MSC/ß-TCP than in those treated with ß-TCP only (p = 0.000). Patients with MSC/ß-TCP implants had better functional recovery (p = 0.028). Conclusion: MSC/ß-TCP prepared by SECCS were effective in the treatment of bone defects in patients with depressed tibial plateau fractures, promoted bone regeneration and improved joint function recovery.

Bone Mesenchymal Stem Cell-Enriched ß-Tricalcium Phosphate Scaffold Processed by the Screen-Enrich-Combine Circulating System Promotes Regeneration of Diaphyseal Bone Non-Union.

Bone non-union after fracture, considered a therapeutic challenge for orthopedics, always needs a reversion surgery, including autograft transplantation (AGT). However, adverse events related to autograft harvest cannot be ignored. Our group designed a novel system called the bone marrow stem cell Screen-Enrich-Combine Circulating System (SECCS) by seeding mesenchymal stem cells (MSCs) into ß-tricalcium phosphate (ß-TCP) during surgery to thereafter rapidly process bioactive bone implantation. In this retrospective case-control study, 30 non-union patients who accepted SECCS therapy and 20 non-union patients who accepted AGT were enrolled. By SECCS therapy, the MSC-enriched ß-TCP particles were implanted into the non-union gap. During the enrichment procedure, a significant proportion of MSCs were screened and enriched from bone marrow into porous ß-TCP particles, and the cells possessed the capacity for three-line differentiation and were CD90+/CD105+/CD34-/CD45-. Approximately 82.0±10.7% of MSCs were enriched from 60 mL bone marrow without damaging cell viability, and approximately 11,444.0±6,018 MSCs were transplanted per patient. No implant-related infections occurred in any case. After 9 months of follow-up, 27 patients (90%) in the SECCS group acquired clinical union, compared with 18 patients (90%) in the AGT group (clinical union time, P = 0.064), and postoperative radiographic union score at 9 months post-operation was similar between the two groups. In conclusion, the SECCS could concentrate a large proportion of MSCs from bone marrow to acquire enough effective cells for therapy without in vitro cell culture. Bone substitutes processed by SECCS demonstrated encouraging promotion of bone regeneration and showed a satisfactory clinical curative effect for diaphyseal bone non-union, which was non-inferior to AGT.

Mesenchymal stem cells and porous ß-tricalcium phosphate composites prepared through stem cell screen-enrich-combine(-biomaterials) circulating system for the repair of critical size bone defects in goat tibia.

BACKGROUND: Efficacious bone substitute is essential for the treatment of a critical size bone defect. Currently, the bone substitutes commonly used in clinical practice lack osteogenic capacity and the therapeutic efficacy is not ideal. Herein, a novel stem cell screen-enrich-combine(-biomaterials) circulating system (SECCS) was introduced to provide the substitutes with osteogenic ability. The stem cell screening, enrichment, and recombination with substitutes could be integrated during the surgical operation. Using SECCS, the bioactive mesenchymal stem cells (MSCs) and porous ß-tricalcium phosphate (ß-TCP) composites (MSCs/ß-TCP) were rapidly prepared for critical size bone defect repair and validated in goat models of critical size tibia defects. METHODS: Twelve goats with right hind limb tibia defects of 30 mm were randomly divided into two groups: six (the experimental group) were treated with MSCs/ß-TCP prepared by SECCS and the other six goats (the control group) were treated with pure porous ß-TCP. The repair effect was assessed by x-ray, computed tomography (CT), micro-CT, histology and histomorphology 6 months after the operation. In addition, the enrichment efficacy of MSCs and the characteristics of the MSCs/ß-TCP prepared by SECCS were evaluated. RESULTS: The SECCS could compound about 81.3 ± 3.0% of the MSCs in bone marrow to the porous ß-TCP without affecting the cell viability. The average number of MSCs for retransplantation was 27,655.0 ± 5011.6 for each goat from the experimental group. In vitro, satisfactory biocompatibility of the MSCs/ß-TCP was performed, with the MSCs spreading adequately, proliferating actively, and retaining the osteogenic potential. In vivo, the defect repair by MSCs/ß-TCP with good medullary cavity recanalization and cortical remodeling was significantly superior to that of pure porous ß-TCP. CONCLUSIONS: The MSCs/ß-TCP prepared through SECCS demonstrated significant therapeutic efficacy in goat models of critical size bone defect. This provides a novel therapeutic strategy for critical size bone defects caused by severe injury, infection, and bone tumor resection with a high profile of safety, effectiveness, simplicity, and ease.

Roles of miR-186 and PTTG1 in colorectal neuroendocrine tumors.

OBJECTIVES: This work aims to investigate the expression of miRNA-186 in patients with colorectal cancer tissues, blood and feces and its roles in regulating the infiltration and invasion in colorectal cancer. METHODS: Totally 39 patients with surgical resection were included from August 2012 to February 2015 in Jilin Province People's Hospital as the Colorectal Neuroendocrine tumor (CNET). Peripheral blood, stool, and resected tumor tissues with adjacent normal of each patient was collected. In the same period, the blood and stool from 25 patients with hemorrhoids or other non-neoplastic diseases were collected and these samples used as clinical control group. MiR-186 expression and PTTG1 (pituitary tumor-transforming 1) expression were detected by quantitative Real-Time PCR (qRT-PCR). The PTTG1 protein expression in tumor samples were detected by Western Blot, while its expression in blood and stool were detected by Elisa. RESULTS: Compared with the control group, the expression of PTTG1 mRNA and protein was significantly up-regulated in tumor samples, blood, and stool of patients with CNET, while the expression of miR-186 was down-regulated (P < 0.05). CONCLUSION: PTTG1 expression was significantly up-regulated in patients with CNET, which was induced by the down-regulated miR-186. MiR-186 may participate in the regulation of infiltration and invasion in CNET patients through targeting PTTG1 expression.