Single-cell RNA-seq analysis reveals the Wnt/Ca2+ signaling pathway with inflammation, apoptosis in nucleus pulposus degeneration.

BACKGROUND: Increasing studies have shown degeneration of nucleus pulposus cells (NPCs) as an critical part of the progression of intervertebral disc degeneration (IVDD). However, there are relatively few studies on single-cell transcriptome contrasts in human degenerated NPCs. Moreover, differences in Wnt/Ca2+ signaling in human degenerated nucleus pulposus cells have not been elucidated. The aim of this study is to investigate the differential expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans and try to investigate its mechanism. METHODS: We performed bioinformatics analysis using our previously published findings to construct single cell expression profiles of normal and degenerated nucleus pulposus. Then, in-depth differential analysis was used to characterize the expression of Wnt/Ca2+ signaling pathway between normal and degenerated nucleus pulposus cells in humans. RESULTS: The obtained cell data were clustered into five different chondrocytes clusters, which chondrocyte 4 and chondrocyte 5 mainly accounted for a high proportion in degenerated nucleus pulposus tissues, but rarely in normal nucleus pulposus tissues. Genes associated within the Wnt/Ca2+ signaling pathway, such as Wnt5B, FZD1, PLC (PLCB1), CaN (PPP3CA) and NAFATC1 are mainly present in chondrocyte 3, chondrocyte 4 and chondrocyte 5 from degenerated nucleus pulposus tissues. In addition, as a receptor that activates Wnt signaling pathway, LRP5 is mainly highly expressed in chondrocyte 5 of degenerated nucleus pulposus cells. Six genes, ANGPTL4, PTGES, IGFBP3, GDF15, TRIB3 and TNFRSF10B, which are associated with apoptosis and inflammatory responses, and are widespread in chondrocyte 4 and chondrocyte 5, may be closely related to degenerative of nucleus pulposus cells. CONCLUSIONS: Single-cell RNA sequencing revealed differential expression of Wnt/Ca2+ signaling in human normal and degenerated nucleus pulposus cells, and this differential expression may be closely related to the abundance of chondrocyte 4 and chondrocyte 5 in degenerated nucleus pulposus cells. In degenerated nucleus pulposus cells, LRP5 activate Wnt5B, which promotes nucleus pulposus cell apoptosis and inflammatory response by regulating the Wnt/Ca2+ signaling pathway, thereby promoting disc degeneration. ANGPTL4, IGFBP3, PTGES in chondrocyte 4 and TRIB3, GDF15, TNFRSF10B in chondrocyte 5 may play an important role in this process.

Corrigendum: Single-cell RNA sequencing reveals the difference in human normal and degenerative nucleus pulposus tissue profiles and cellular interactions.

[This corrects the article DOI: 10.3389/fcell.2022.910626.].

Single-cell RNA landscape of osteoimmune microenvironment in osteoporotic vertebral compression fracture and Kümmell's disease.

Background: Single-cell RNA sequencing (scRNA-seq) enables specific analysis of cell populations at single-cell resolution; however, there is still a lack of single-cell-level studies to characterize the dynamic and complex interactions between osteoporotic vertebral compression fractures (OVCFs) and Kümmell's disease (KD) in the osteoimmune microenvironment. In this study, we used scRNA-seq analysis to investigate the osteoimmune microenvironment and cellular composition in OVCFs and KD. Methods: ScRNA-seq was used to perform analysis of fractured vertebral bone tissues from one OVCF and one KD patients, and a total of 8,741 single cells were captured for single-cell transcriptomic analysis. The cellularity of human vertebral bone tissue was further analyzed using uniform manifold approximation and projection. Pseudo-time analysis and gene enrichment analysis revealed the biological function of cell fate and its counterparts. CellphoneDB was used to identify the interactions between bone cells and immune cells in the osteoimmune microenvironment of human vertebral bone tissue and their potential functions. Results: A cellular profile of the osteoimmune microenvironment of human vertebral bone tissue was established, including mesenchymal stem cells (MSCs), pericytes, myofibroblasts, fibroblasts, chondrocytes, endothelial cells (ECs), granulocytes, monocytes, T cells, B cells, plasma cells, mast cells, and early erythrocytes. MSCs play an immunoregulatory function and mediate osteogenic differentiation and cell proliferation. The differentiation trajectory of osteoclasts in human vertebral bone tissue was also revealed. In addition, ECs actively participate in inflammatory infiltration and coupling with bone cells. T and B cells actively participate in regulating bone homeostasis. Finally, by identifying the interaction of ligand-receptor pairs, we found that immune cells and osteoclasts have bidirectional regulatory characteristics, have the effects of regulating bone resorption by osteoclasts and promoting bone formation, and are essential for bone homeostasis. It is also highlighted that CD8-TEM cells and osteoclasts might crosstalk via CD160-TNFRSF14 ligand-receptor interaction. Conclusion: Our analysis reveals a differential landscape of molecular pathways, population composition, and cell-cell interactions during OVCF development into KD. OVCFs exhibit a higher osteogenic differentiation capacity, owing to abundant immune cells. Conversely, KD results in greater bone resorption than bone formation due to depletion of MSCs and a relatively suppressed immune system, and this immune imbalance eventually leads to vertebral avascular necrosis. The site of action between immune cells and osteoclasts is expected to be a new therapeutic target, and these results may accelerate mechanistic and functional studies of osteoimmune cell types and specific gene action in vertebral avascular necrosis and pathological bone loss diseases, paving the way for drug discovery.

Corrigendum: Single-cell RNA sequencing reveals the difference in human normal and degenerative nucleus pulposus tissue profiles and cellular interactions.

[This corrects the article DOI: 10.3389/fcell.2022.910626.].

Single-Cell RNA Sequencing Reveals the Difference in Human Normal and Degenerative Nucleus Pulposus Tissue Profiles and Cellular Interactions.

Background: The nucleus pulposus is a constituent structure of the human intervertebral disc, and its degeneration can cause intervertebral disc degeneration (IDD). However, the cellular and molecular mechanisms involved remain elusive. Methods: Through bioinformatics analysis, the single-cell transcriptome sequencing expression profiles of human normal nucleus pulposus (NNP) cells and human degenerative nucleus pulposus (DNP) cells were compared to clarify the transcriptome differential expression profiles of human NNP and DNP. The single-cell sequencing results of the two samples were analyzed using bioinformatics methods to compare the differences in histiocytosis between human NNP and DNP, map the histiocytes of NNP and DNP, perform cell differentiation trajectories for the cell populations of interest and predict cell function, and explore their heterogeneity by pathway analysis and Gene Ontology analysis. Results: Nine cell types were identified, which were chondrocyte 1, chondrocyte 2, chondrocyte 3, chondrocyte 4, chondrocyte 5, endothelial, macrophage, neutrophil, and T cells. Analysis of the proportion of chondrocytes in different tissues revealed that chondrocyte 1 accounted for a higher proportion of NNP cells and highly expressed COL2A1 compared with DNP cells; chondrocyte 2, chondrocyte 3, chondrocyte 4, and chondrocyte 5 accounted for a higher proportion of DNP cells compared with NNP cells. Among them, chondrocyte 2 was an inhibitory calcified chondrocyte with high expression of MGP, chondrocytes 3 were fibrochondrocytes with high expression of COL1A1, chondrocytes 4 were chondrocytes that highly express pain inflammatory genes such as PTGES, and chondrocytes 5 were calcified chondrocytes with high expression of FN1 (chondrocytes 4 and chondrocytes 5 were found for the first time in a study of single-cell transcriptome sequencing of disc tissue). Cell trajectory analysis revealed that chondrocyte 1 was at the beginning of the trajectory and chondrocyte 3 was at the end of the trajectory, while chondrocyte 5 appeared first in the trajectory relative to chondrocyte 2 and chondrocyte 4. Conclusion: After functional identification of the specifically expressed genes in five chondrocytes, it was found that chondrocyte 1 was a chondrocyte with high expression of COL2A1, COL9A2, COL11A2, and CHRDL2 in a high proportion of NNP cells, and chondrocyte 3 was a fibrochondrocyte with high expression of COL1A1, COL6A3, COL1A2, COL3A1, AQP1, and COL15A1 in an increased proportion during nucleus pulposus cell degeneration. Through cell trajectory analysis, it was found that chondrocytes 5 specifically expressing FN1, SESN2, and GDF15 may be the key cells leading to degeneration of nucleus pulposus cells. Chondrocytes 2 expressing MGP, MT1G, and GPX3 may play a role in reversing calcification and degeneration, and chondrocytes 4 expressing PTGES, TREM1, and TIMP1 may play a role in disc degeneration pain and inflammation.

Expression of Matrix Metalloprotein 13 in Injury Model of Articular Chondrocyte in Rabbits and Analysis of Nano-Drug-Loading System.

This study aimed to analyze the application of a responsive nano-drug-loading system in injury model of articular chondrocyte in rabbits, as well as its effect on expression of matrix metalloprotein 13 (MMP13). The nanoprecipitation method was adopted to prepare camptothecin (CPT)-loaded poly ethylene glycol (PEG)-Poly caprolactone (PCL) and PEG-PCL nanoparticles without CPT. Afterward, the above mentioned nano-drug-loaded system was used to treat an in vitro scratch model of articular chondrocytes. According to different treatment plans, they were divided into groups: G0 (administered CPT-PEG-PCL nanomedicine), G1 (administered PEG-PCL drug), G2 (saline control), and G3 (healthy control). Results showed that the drug-loading capacity and efficiency of CPT-PEG-PCL was higher than that of PEG-PCL. The levels of type II collagen and hyaluronic acid in G0 was higher than that in G1 and G2. The levels of type II collagen and hyaluronic acid in G0 were not obviously different from those in G3. The level of MMP13 in G0 was lower than that in G1 and G2 and the level of tissue inhibitor of metalloproteinases 1 (TIMP1) in G0 was higher than that in G1 and G2. The proliferation activity of cells in G0 was higher than that in G1 and G2, but there was no obvious difference when compared with G3. In conclusion, CPT-PEG-PCL has stronger long-term circulation capacity and drug-loading efficiency. It can effectively up-regulate the levels of type II collagen, hyaluronic acid, and TIMP1, as well as reduce the synthesis and secretion of MMP13 and promote the repair of articular cartilage damage.

Analysis of different hematoma expansion shapes caused by different risk factors in patients with hypertensive intracerebral hemorrhage.

OBJECTIVE: To elucidate the relationship between the risk factors and hematoma expansion(HE)shapes. PATIENTS AND METHODS: From February 2013 to November 2018, 60 patients diagnosed as basal ganglia ICH were divided into the filled type hematoma expansion group (FTE group) and the expanded type hematoma expansion group (ETE group). we performed follow-up CT and three-dimensional reconstruction for the patients and compared the hematoma before and after the expansion of size and extent. RESULTS: The regression analysis showed that the irregular sign (odds ratio, 3.64; 95 % CI, 1.46-9.12), black hole sign (odds ratio, 3.85; 95 % CI, 1.40-10.60), blend sign (odds ratio, 2.86; 95 % CI, 1.03-7.95), and early use of dehydration (odds ratio, 4.59; 95 % CI, 1.59-13.19) were possible risk factors for the ETE group, while the high systolic blood pressure (odds ratio, 1.51; 95 % CI, 1.04-2.30), early use of dehydration (odds ratio, 3.27; 95 % CI, 1.10-9.69) and low density low-density band (odds ratio, 4.52; 95 % CI, 1.54-13.28) were possible risk factors for the FTE group. CONCLUSIONS: The irregular sign, black hole sign, blend sign and early use of dehydration may be the main risk factors for ETE, whereas early use of dehydration, high systolic blood pressure, and low density low-density band may be the main risk factors for FTE.

Hemoglobin Concentration Affects Hypertensive Basal Ganglia Hemorrhage After Surgery: Correlation Analysis in a High-Altitude Region.

OBJECTIVE: Hypertensive cerebral hemorrhage leads to greater mortality and worse functional outcomes at high altitudes. Experimental studies have suggested that hemoglobin can lead to increased perihemorrhagic edema after intracerebral hemorrhage. METHODS: Patients were divided into a high-hemoglobin (H-H) group (>180 g/L) and a low-hemoglobin (L-H) group (≤180 g/L). The distance from the cortex to the midline was used to indicate the degree of edema. At 1, 7, 14, and 21 days, the patients' status was scored using the Glasgow coma scale, and survival was plotted using Kaplan-Meier survival curves. Pearson correlation analysis showed that the difference between the postoperative and preoperative Glasgow coma scale score correlated with the hemoglobin concentration. The Glasgow outcome scale was used to assess neurological recovery after 6 months. RESULTS: On days 7, 14, and 21, the edema of the H-H group was significantly greater than that of the L-H group (P < 0.01 and P < 0.001, respectively). The edema of the H-H group peaked at 14 and 21 days, but that of the L-H group peaked at 7 days. The hemoglobin concentration and postoperative neurological recovery had a linear relationship in the H-H group. The L-H group had greater survival compared with the H-H group (P < 0.05). The L-H group had higher Glasgow outcome scale scores compared with the H-H group (P < 0.05). CONCLUSION: The hemoglobin concentration affects the mortality and morbidity from hypertensive cerebral hemorrhage in high-altitude regions, and a linear relationship exists between hemoglobin concentration and neurological recovery in the H-H group.

[Mid-term effectiveness of Coflex interspinous dynamic internal fixation combined with spinal fusion for lumbar disc degeneration].

Objective: To evaluate the effectiveness of Coflex interspinous dynamic internal fixation combined with spinal fusion for lumbar disc degeneration. Methods: The clinical data of 39 patients with two-level lumbar disc degeneration who met the selection criteria between June 2010 and December 2011 was retrospectively analyzed. They were divided into group A (20 cases, simple lumbar decompression and fusion) and group B (19 cases, Coflex interspinous dynamic internal fixation combined with spinal fusion) according to different surgical methods. There was no significant difference in age, gender, disease diagnosis, lesion segment, disease duration, Oswestry disability index (ODI), visual analogue scale (VAS) score, and the intervertebral height, foramen intervertebral height (FIH), and range of motion (ROM) of upper operative segment and adjacent segment between the two groups ( P>0.05). ODI and VAS score were used to evaluate the effectiveness before operation and at last follow-up, and the improvement rates were calculated. The intervertebral height [anterior disc height (ADH), middle disc height (MDH), and posterior disc height (PDH)], FIH, and ROM were measured and compared between the two groups. Results: The operation time and intraoperative blood loss in group A were significantly more than those in group B ( P<0.05), and there was no significant difference in hospitalization time between the two groups ( t=0.992, P=0.328). All patients were followed up; the follow-up time was 33-50 months (mean, 40.5 months) in group A and 39-51 months (mean, 42.6 months) in group B. No complication such as displacement, loosening, or rupture of internal fixator was found in both groups. At last follow-up, ODI and VAS score of the two groups significantly improved when compared with preoperative scores ( P<0.05). At last follow-up, there was no significant difference in ODI, VAS score, and improvement rate of ODI between the two groups ( P>0.05); the improvement rate of VAS score in group B was significantly higher than that in group A ( t=2.245, P=0.031). There was no significant difference in the intervertebral height and FIH of the upper operative segment at last follow-up between the two groups and between preoperation and last follow-up in the two groups ( P>0.05). At last follow-up, the ADH of adjacent segment in group B was significantly higher than that in group A, and MDH, PDH, and FIH were significantly lower than those in group A ( P<0.05). Compared with preoperation, the ADH of adjacent segment in group A decreased and MDH, PDH, and FIH increased at last follow-up ( P<0.05), while all indexes in group B did not change significantly ( P>0.05). The ROM of adjacent segment in group A increased significantly at last follow-up ( t=2.318, P=0.026). There was significant difference in ROM of adjacent segment between the two groups ( P<0.05). Conclusion: The mid-term effectiveness of Coflex interspinous dynamic internal fixation combined with spinal fusion is similar to that of simple decompression fusion. For those patients whose adjacent segments of the responsible segments have degeneration but have no symptoms or mild symptoms, this treatment can slow down the adjacent segment degeneration.

Corrigendum to "Moderate Fluid Shear Stress Could Regulate the Cytoskeleton of Nucleus Pulposus and Surrounding Inflammatory Mediators by Activating the FAK-MEK5-ERK5-cFos-AP1 Signaling Pathway".

[This corrects the article DOI: 10.1155/2018/9405738.].

Moderate Fluid Shear Stress Could Regulate the Cytoskeleton of Nucleus Pulposus and Surrounding Inflammatory Mediators by Activating the FAK-MEK5-ERK5-cFos-AP1 Signaling Pathway.

We first applied moderate fluid shear stress to nucleus pulposus cells. The correlation of AP-1 with type II collagen, proteoglycan, Cytokeratin 8 protein, MAP-1, MAP-2, and MAP-4 and the correlation of AP-1 with IL-1ß, TNF-α, IL-6, IL-8, MIP-1, MCP-1, and NO were detected. Our results document that moderate fluid shear stress could activate the FAK-MEK5-ERK5-cFos-AP1 signaling pathway. AP1 could downregulate the construct factors of cytoskeleton such as type II collagen, proteoglycan, Cytokeratin 8 protein, MAP-1, MAP-2, and MAP-4 in nucleus pulposus cell after the fluid shear stress was loaded. AP1 could upregulate the inflammatory factors such as IL-1ß, TNF-α, IL-6, IL-8, MIP-1, MCP-1, and NO in nucleus pulposus cell after the fluid shear stress was loaded. Taken together, our data suggested that moderate fluid shear stress may play an important role in the cytoskeleton of nucleus pulposus and surrounding inflammatory mediators by activating the FAK-MEK5-ERK5-cFos-AP1 signaling pathway, thereby affecting cell degeneration.

A case of Galen vein thrombosis occurring after bilateral acetabular fractures in the Tibet plateau - what can we learn?

Hypoxia leads to increased red blood cells and blood viscosity at high altitude while moderate trauma increases coagulation in blood. Under the above-mentioned conditions, venous sinus thrombosis is more likely to occur. A patient suffering bilateral acetabular fractures together with the gradual disturbance of consciousness was admitted to our hospital. Though computed tomography arteriogram (CTA) of the brain displayed normal blood vessels; bilateral thalamus and brainstem infarction were found on head computed tomography (CT) and Galen vein thrombosis on cerebral computed tomography venography (CTV). Dehydration and tracheotomy were immediately conducted with antiplatelet, anticoagulant and neurotrophic medicine administered to the patient. After three days' treatment, the patient's consciousness gradually improved and eventually became clear enough to leave the hospital. On follow-up, no dysfunction was documented.

Protective effect of p53 on the viability of intervertebral disc nucleus pulposus cells under low glucose condition.

P53 is a famous cancer suppressor and plays key roles in metabolism. Intervertebral disc (IVD) is the largest avascular cartilaginous structure in humans and its degeneration is a common cause of spine diseases initiated from damaged nucleus pulposus (NP) cells. The potential cause of disc degeneration has been attributed to aging, genetic factors, mechanical factors and nutrition. In this study, we found that p53 decreased and leaked to the cytoplasm in NP cells as the glucose level decreases, in contrast to cancer cells in which p53 increases and concentrates to the nuclei. Comparing with in p53 knockdown NP cells, relative high p53 expression in normal control NP cells inhibited autophagy and the pentose phosphate pathway. Furthermore, the expression of Sox 9 and type II collagen were higher in p53 normal control than p53 knockdown NP cells. Based on these results, we believe that relative high p53 facilitates NP cell viability and integrity.

miR-155 Inhibits Nucleus Pulposus Cells' Degeneration through Targeting ERK 1/2.

We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2.

Fuse-binding protein 1 is a target of the EZH2 inhibitor GSK343, in osteosarcoma cells.

Osteosarcoma is the primary cancer of leaf tissue and is regarded as a differentiation disease caused by genetic and epigenetic changes which interrupt the osteoblast differentiation from mesenchymal stem cells. Because of its high malignancy degree and rapid development, the morbidity and mortality are high. The enhancer of zeste homolog 2 (EZH2) is a catalytic subunit of polycomb repressive complex 2 (PRC2) and has been demonstrated to be involved in a variety of biological processes, such as cell proliferation and program cell death. EZH2 impairs gene expression by catalyzing the tri-methylation of histone H3 lysine 27 (H3K27me3) which controls gene transcription epigenetically. It is reported that EZH2 expression is higher in osteosarcoma than in osteoblastoma and the highest expression of EZH2 is found in osteosarcoma with metastasis. In the past few years, several potent inhibitors of EZH2 have been discovered, and GSK343 is one of them. In this study, we found that GSK343 inhibited osteosarcoma cell viability, restrained cell cycle transition and promoted programmed cell death. GSK343 not only inhibited the expression of EZH2 and its target, c-Myc and H3K27me3, but it also inhibited fuse binding protein 1 (FBP1) expression, another c-Myc regulator. Furthermore, we found that FBP1 physically interacts with EZH2. Based on these results, we believe that GSK343 is a potential molecule for osteosarcoma clinical treatment. Other than the inhibition on EZH2-c-Myc signal pathway, we postulate that the inhibition on FBP1-c-Myc signal pathway is another potential underlying mechanism with which GSK343 inhibits osteosarcoma cell viability.

Elevated Hemoglobin Concentration Affects Acute Severe Head Trauma After Recovery from Surgery of Neurologic Function in the Tibetan Plateau.

BACKGROUND: Long-lived inhabitants of the plateau region have a higher hemoglobin concentration, which is the leading cause of damage in various organs, especially the nervous system. The aim of this study was to investigate the effect of hemoglobin concentration on mortality and neural functions after decompressive craniectomy in patients with hypoxia. METHODS: Patients with acute severe head trauma were classified into an elevated hemoglobin concentration group (EHb) and a moderate hemoglobin concentration group (MHb). The survival rate was evaluated by comparing the Glasgow Coma Score on days 1, 3, 7, and 15 after surgery (Kaplan-Meier survival curve). The Glasgow Outcome Scale classification method was used to evaluate recovery of neural function. RESULTS: The Glasgow Coma Score scores on days 3, 7, and 15 were lower in the EHb group compared with the MHb group (P < 0.05). Mortality was significantly higher in the EHb group compared with the MHb group (P < 0.05). After 6 months, the Glasgow Outcome Scale of the MHb group was significantly higher than that of the EHb group (P < 0.05). CONCLUSIONS: Elevated hemoglobin concentration has a serious impact on neurologic recovery and mortality, as seen in patients with acute severe head trauma after decompressive craniectomy.

Natural Germacrane Sesquiterpenes Inhibit Osteoclast Formation, Bone Resorption, RANKL-Induced NF-κB Activation, and IκBα Degradation.

Osteolytic bone diseases are commonly presented with enhanced osteoclast formation and bone resorption. Sesquiterpene lactone natural compounds have been found to possess anti-inflammatory and immune-modulation effects. Here, we identified three germacrane sesquiterpenes using computer-based virtual screening for the structural similarity with sesquiterpene lactone, parthenolide. We showed that natural germacrane sesquiterpene compounds A, B, and C inhibit osteoclast formation and bone resorption in a dose-dependent manner, with relative potency compound A > compound C > compound B based on their equimolar concentrations. Mechanistic studies by Luciferase reporter gene assay and Western blot analysis showed that germacrane sesquiterpene compound A inhibits RANKL-induced activation of NF-κB and IκBα degradation. This study reveals that natural germacrane sesquiterpene compounds are inhibitors for osteoclast formation and bone resorption, and provides evidence that naturally-occurring compounds might be beneficial as alternative medicine for the prevention and treatment of osteolysis.

[EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR a AND SERUM DEPRIVATION].

OBJECTIVE: To explore the effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor a (TNF-alpha) and serum deprivation. METHODS: The NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups: Vit C group (group A), TNF-alpha group (group B), and serum deprivation group (group C). Group A was reassigned to Al subgroup (basic medium), A2 subgroup (100 pg/mL Vit C), and A3 subgroup (200 pg/mL Vit C). Group B was reassigned to B0 subgroup (control group), Bi subgroup (100 ng/mL TNF-alpha), B2 subgroup (100 microg/mL Vit C+100 ng/mL TNF-alpha), and B3 subgroup (200 microg/mL Vit C+100 ng/mL TNF-alpha). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2% FBS+100 microg/mL Vit C), and C3 subgroup (2% FBS+200 microg/mL Vit C). After application of 100 pg/mL or 200 microg/mL Vit C for 24 hours, NP cells were stimulated by TNF-alpha and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type I, collagen type II, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR. Results Group A: Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with Al subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type I, collagen type II, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B: TNF-alpha significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C: 2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05). CONCLUSION: Vit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 microg/mL Vit C may delay the apoptosis induced by TNF-alpha and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.

Effect of Osteonecrosis Intervention Rod Versus Core Decompression Using Multiple Small Drill Holes on Early Stages of Necrosis of the Femoral Head: A Prospective Study on a Series of 60 Patients with a Minimum 1-Year-Follow-Up.

INTRODUCTION: The conventional CD used 10 mm drill holes associated with a lack of structural support. Thus, alternative methods such as a tantalum implant, small drill holes, and biological treatment were developed to prevent deterioration of the joint. The treatment of CD by multiple 3.2 mm drill holes could reduce the femoral neck fracture and partial weight bearing was allowed. This study was aimed to evaluate the effect of osteonecrosis intervention rod versus core decompression using multiple small drill holes on early stages of necrosis of the femoral head. METHOD: From January 2011 to January 2012, 60 patients undergoing surgery for osteonecrosis with core decompression were randomly assigned into 2 groups based on the type of core decompression used: (1) a total of 30 osteonecrosis patients (with 16 hips on Steinburg stageⅠ,20 hips on Steinburg stageⅡ) were treated with a porous tantalum rod insertion. The diameter of the drill hole for the intervention rod was 10mm.(2) a total of 30 osteonecrosis patients (with 14 hips on Steinburg stageⅠ,20 hips on Steinburg stageⅡ) were treated with core decompression using five drill holes on the lateral femur, the diameter of the hole was 3.2 mm. The average age of the patient was 32.6 years (20-45 years) and the average time of follow-up was 25.6 months (12- 28 months) in the rod implanted group. The average age of the patient was 35.2 years (22- 43 years) and the average time of follow-up was 26.3 months (12-28 months) in the small drill holes group. RESULTS: The average of surgical time was 40 min, and the mean volume of blood loss was 30 ml in both surgical groups. The average of Harris score was improved from 56.2 ± 7.1 preoperative to 80.2 ± 11.4 at the last follow-up in the rod implanted group (p < 0.05). The mean Harris score was improved from 53.8 ± 6.6 preoperative to 79.7 ± 13.2 at the last follow-up in the small drill holes group (p<0. 05). No significant difference was observed in Harris score between the two groups. At the last follow-up, 28 of 36 hips were at the same radiographic stages as pre-operation, and 8 deteriorated in the rod implanted group. 26 of 34 hips were at the same radiographic stage as pre-operation, and 8 deteriorated in the small drill holes group. No significant difference was observed in radiographic stage between the two groups. There was no favourable result on the outcome of a tantalum intervention implant compared to multiple small drill holes. DISCUSSION: CD via multiple small drill holes would allow similar postoperative load-bearing and seems to result in similar or even better clinical outcome without the prolonged implantation of an expensive tantalum implant. A tantalum rod intervention and core decompression using multiple small drill holes were effective on the stage I hips rather than stage II hips.

Preparation and characterization of a novel injectable strontium-containing calcium phosphate cement with collagen.

PURPOSE: To develop a novel injectable strontium-containing calcium phosphate cement with collagen. METHODS: A novel calcium phosphate bone cement (CPC) was prepared with the addition of strontium element, collagenl, and modified starch; the injectability, solidification time, microstructure, phase composition, compressive strength, anti-collapsibility and histological properties of material were evaluated. RESULTS: The results showed that the material could be injected with an excellent performance; the modified starch significantly improved the anti-washout property of cement; with the liquid to solid ratio of 0.3, the largest compressive strength of cement was obtained (48.0 MPa ± 2.3 MPa); histological examination of repair tissue showed that the bone was repaired after 16 weeks; the degradation of cement was consistent with the new bone growth. CONCLUSION: A novel injectable collagen-strontium-containing CPC with excellent compressive strength and suitable setting time was prepared, with addition of modified starch. The CPC showed a good anti-washout property and the degradation time of the cement met with the new bone growing. This material is supposed to be used in orthopedic and maxillofacial surgery for bone defects.