PHLDA2 overexpression facilitates senescence and apoptosis via the mitochondrial route in human nucleus pulposus cells by regulating Wnt/ß-catenin signalling pathway.

Low back pain is a common clinical symptom of intervertebral disc degeneration (IVDD), which seriously affects the quality of life of the patients. The abnormal apoptosis and senescence of nucleus pulposus cells (NPCs) play important roles in the pathogenesis of IVDD. PHLDA2 is an imprinted gene related to cell apoptosis and tumour progression. However, its role in NPC degeneration is not yet clear. Therefore, this study was set to explore the effects of PHLDA2 on NPC senescence and apoptosis and the underlying mechanisms. The expression of PHLDA2 was examined in human nucleus pulposus (NP) tissues and NPCs. Immunohistochemical staining, magnetic resonance imaging imaging and western blot were performed to evaluate the phenotypes of intervertebral discs. Senescence and apoptosis of NPCs were assessed by SA-ß-galactosidase, flow cytometry and western blot. Mitochondrial function was investigated by JC-1 staining and transmission electron microscopy. It was found that the expression level of PHLDA2 was abnormally elevated in degenerated human NP tissues and NPCs. Furthermore, knockdown of PHLDA2 can significantly inhibit senescence and apoptosis of NPCs, whereas overexpression of PHLDA2 can reverse senescence and apoptosis of NPCs in vitro. In vivo experiment further confirmed that PHLDA2 knockdown could alleviate IVDD in rats. Knockdown of PHLDA2 could also reverse senescence and apoptosis in IL-1ß-treated NPCs. JC-1 staining indicated PHLDA2's knockdown impaired disruption of the mitochondrial membrane potential and also ameliorated superstructural destruction of NPCs as showed by transmission electron microscopy. Finally, we found the PHLDA2 knockdown promoted Collagen-II expression and suppressed MMP3 expression in NPCs by repressing wnt/ß-catenin pathway. In conclusion, the results of the present study showed that PHLDA2 promotes IL-1ß-induced apoptosis and senescence of NP cells via mitochondrial route by activating the Wnt/ß-catenin pathway, and suggested that therapy targeting PHLDA2 may provide valuable insights into possible IVDD therapies.

Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt.

The accumulation of metabolites in the intervertebral disc is considered an important cause of intervertebral disc degeneration (IVDD). Lactic acid, which is a metabolite that is produced by cellular anaerobic glycolysis, has been proven to be closely associated with IVDD. However, little is known about the role of lactic acid in nucleus pulposus cells (NPCs) senescence and oxidative stress. The aim of this study was to investigate the effect of lactic acid on NPCs senescence and oxidative stress as well as the underlying mechanism. A puncture-induced disc degeneration (PIDD) model was established in rats. Metabolomics analysis revealed that lactic acid levels were significantly increased in degenerated intervertebral discs. Elimination of excessive lactic acid using a lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentrations of lactic acid could induce senescence and oxidative stress in NPCs. High-throughput RNA sequencing results and bioinformatic analysis demonstrated that the induction of NPCs senescence and oxidative stress by lactic acid may be related to the PI3K/Akt signaling pathway. Further study verified that high concentrations of lactic acid could induce NPCs senescence and oxidative stress by interacting with Akt and regulating its downstream Akt/p21/p27/cyclin D1 and Akt/Nrf2/HO-1 pathways. Utilizing molecular docking, site-directed mutation and microscale thermophoresis assays, we found that lactic acid could regulate Akt kinase activity by binding to the Lys39 and Leu52 residues in the PH domain of Akt. These results highlight the involvement of lactic acid in NPCs senescence and oxidative stress, and lactic acid may become a novel potential therapeutic target for the treatment of IVDD.

Aberrant mechanical loading induces annulus fibrosus cells apoptosis in intervertebral disc degeneration via mechanosensitive ion channel Piezo1.

BACKGROUND: Intervertebral disc degeneration (IVDD) is closely associated with the structural damage in the annulus fibrosus (AF). Aberrant mechanical loading is an important inducement of annulus fibrosus cells (AFCs) apoptosis, which contributes to the AF structural damage and aggravates IVDD, but the underlying mechanism is still unclear. This study aims to investigate the mechanism of a mechanosensitive ion channel protein Piezo1 in aberrant mechanical loading-induced AFCs apoptosis and IVDD. METHODS: Rats were subjected to lumbar instability surgery to induce the unbalanced dynamic and static forces to establish the lumbar instability model. MRI and histological staining were used to evaluate the IVDD degree. A cyclic mechanical stretch (CMS)-stimulated AFCs apoptosis model was established by a Flexcell system in vitro. Tunel staining, mitochondrial membrane potential (MMP) detection, and flow cytometry were used to evaluate the apoptosis level. The activation of Piezo1 was detected using western blot and calcium fluorescent probes. Chemical activator Yoda1, chemical inhibitor GSMTx4, and a lentiviral shRNA-Piezo1 system (Lv-Piezo1) were utilized to regulate the function of Piezo1. High-throughput RNA sequencing (RNA-seq) was used to explore the mechanism of Piezo1-induced AFCs apoptosis. The Calpain activity and the activation of Calpain2/Bax/Caspase3 axis were evaluated by the Calpain activity kit and western blot with the siRNA-mediated Calapin1 or Calpain2 knockdown. Intradiscal administration of Lv-Piezo1 was utilized to evaluate the therapeutic effect of Piezo1 silencing in IVDD rats. RESULTS: Lumbar instability surgery promoted the expression of Piezo1 in AFCs and stimulated IVDD in rats 4 weeks after surgery. CMS elicited distinct apoptosis of AFCs, with enhanced Piezo1 activation. Yoda1 further promoted CMS-induced apoptosis of AFCs, while GSMTx4 and Lv-Piezo1 exhibited opposite effects. RNA-seq showed that knocking down Piezo1 inhibited the calcium signaling pathway. CMS enhanced Calpain activity and elevated the expression of BAX and cleaved-Caspase3. Calpain2, but not Calpain1 knockdown, inhibited the expression of BAX and cleaved-Caspase3 and alleviated AFCs apoptosis. Lv-Piezo1 significantly alleviated the progress of IVDD in rats after lumbar instability surgery. CONCLUSIONS: Aberrant mechanical loading induces AFCs apoptosis to promote IVDD by activating Piezo1 and downstream Calpain2/BAX/Caspase3 pathway. Piezo1 is expected to be a potential therapeutic target in treating IVDD.

Optineurin-mediated mitophagy as a potential therapeutic target for intervertebral disc degeneration.

Low back pain is thought to be mainly caused by intervertebral disc degeneration (IVDD), and there is a lack of effective treatments. Cellular senescence and matrix degradation are important factors that cause disc degeneration. Mitochondrial dysfunction induced by oxidative stress is an important mechanism of cellular senescence and matrix degradation in the nucleus pulposus (NP), and mitophagy can effectively remove damaged mitochondria, restore mitochondrial homeostasis, and mitigate the damage caused by oxidative stress. Optineurin (OPTN) is a selective mitophagy receptor, and its role in intervertebral disc degeneration remains unclear. Here, we aimed to explore the effect of OPTN on H2O2-induced nucleus pulposus cell (NPCs) senescence and matrix degradation in a rat model of disc degeneration. Western blot analysis showed that OPTN expression was reduced in degenerative human and rat nucleus pulposus tissues and increased in H2O2-induced senescent NPCs. OPTN overexpression significantly inhibited H2O2-induced senescence and increased matrix-associated protein expression in NPCs, but OPTN knockdown showed the opposite effect. As previous reports have suggested that mitophagy significantly reduces mitochondrial damage and reactive oxygen species (ROS) caused by oxidative stress, and we used the mitophagy agonist CCCP, the mitophagy inhibitor cyclosporin A (CsA), and the mitochondrial ROS (mtROS) scavenger mitoTEMPO and confirmed that OPTN attenuated NPCs senescence and matrix degeneration caused by oxidative stress by promoting mitophagy to scavenge damaged mitochondria and excess reactive oxygen species, thereby slowing the progression of IVDD. In conclusion, our research suggests that OPTN is involved in IVDD and exerts beneficial effects against IVDD.

Pretreatment of nucleus pulposus mesenchymal stem cells with appropriate concentration of H2O2 enhances their ability to treat intervertebral disc degeneration.

BACKGROUND: Nucleus pulposus mesenchymal stem cells (NPMSCs) transplantation is a promising treatment for intervertebral disc degeneration (IVDD). However, the transplanted NPMSCs exhibited weak cell proliferation, high cell apoptosis, and a low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need to explore feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation. OBJECTIVE: To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (H2O2) and explore the therapeutic efficacy of NPMSCs transplantation using H2O2 pretreatment in IVDD. METHODS: Rat NPMSCs were pretreated with different concentrations (range from 25 to 300 µM) of H2O2. The proliferation, reactive oxygen species (ROS) level, and apoptosis of NPMSCs were detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and flow cytometry in vitro. The underlying signalling pathways were explored utilizing Western blotting. A rat needle puncture-stimulated IVDD model was established. X-ray, histological staining, and a multimode small animal live imaging system were used to evaluate the therapeutic effect of H2O2-pretreated NPMSCs in vivo. RESULTS: NPMSCs pretreated with 75 µM H2O2 demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 µM H2O2-pretreated NPMSCs exhibited significantly enhanced antioxidative stress ability (P < 0.01), which is related to downregulated Brd4 and Keap1 and upregulated Nrf2. NPMSCs pretreated with 75 µM H2O2 also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 µM H2O2-pretreated NPMSCs-transplanted rats exhibited an enhanced disc height index (DHI% = 90.00 ± 4.55, P < 0.01) and better histological morphology (histological score = 13.5 ± 0.5, P < 0.01), which means 75 µM H2O2-pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD. CONCLUSIONS: Pretreatment with 75 µM H2O2 was the optimal concentration to improve the proliferation, antioxidative stress, and antiapoptotic ability of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.

Retrospective Comparative Study of Pedicle Screw Fixation via Quadrant Retractor and Buck's Technique in the Treatment of Adolescent Spondylolysis.

OBJECTIVE: To compare the effectiveness and practicality of pedicle screw fixation via the Quadrant retractor and Buck's technique in the treatment of adolescent spondylolysis. METHODS: A total of 31 patients who underwent pedicle screw fixation or Buck's technique at our hospital from 2012 to 2017 were selected for this retrospective study. The patients were divided into a pedicle screw group (16 patients) and a Buck's technique group (15 patients) according to surgical procedure. Age, sex, disease duration, involved segments, preoperative Oswestry disability index (ODI) scores, visual analogue scale (VAS) scores for low back pain (LBP), intraoperative blood loss, incision length, operative time and length of hospital stay were documented. ODI scores, VAS scores for LBP and fusion rates at 1 month, 6 months, 1 year and 3 years postoperatively were used to evaluate surgical outcomes. RESULTS: The average follow-up period was 32.75 ± 11.99 months in the pedicle screw group and 31.02 ± 9.64 months in the Buck's technique group. No significant differences in demographic data and perioperative data were found between the two groups (P > 0.05). The ODI scores and VAS scores for LBP in both groups were significantly improved at 3 years postoperatively compared with the values before surgery (ODI%: 45.74 ± 2.47 vs 10.99 ± 3.00; 45.29 ± 6.94 vs 15.73 ± 6.89. VAS: 5.94 ± 0.68 vs 1.50 ± 0.52; 6.13 ± 0.74 vs 2.13 ± 0.92, P < 0.05). The ODI scores of the patients in the pedicle screw group at 1 month to 3 years postoperatively were lower than those of the patients in the Buck's technique group (P < 0.05). Moreover, the VAS scores for LBP of the patients in the pedicle screw group at 6 months and 3 years postoperatively were lower than those of the patients in the Buck's technique group (P < 0.05). No significant difference in the VAS scores for LBP was found between the two groups at 1 month postoperatively (3.88 ± 0.50 vs 4.20 ± 0.56, P = 0.10). Three years postoperatively, good fusion of the pars interarticularis was achieved in all patients in the pedicle screw group, but four patients in the Buck's technique group did not achieve good fusion (P = 0.02). CONCLUSION: Both pedicle screw fixation and Buck's technique can achieve good outcomes in the treatment of adolescent spondylolysis. Pedicle screw fixation via the Quadrant retractor for the treatment of spondylolysis is associated with more satisfactory effects in terms of LBP relief and fusion results.

The biological effect of cobalt chloride mimetic-hypoxia on nucleus pulposus cells and the comparability with physical hypoxia in vitro.

Objective: Nucleus pulposus cells (NPCs) are cells extracted from the intervertebral disc and are important for research into intervertebral disc degeneration (IVDD). NPCs live in an avascular and relatively hypoxic environment. Cobalt chloride (CoCl2) has been used in many cell studies to mimic hypoxia. The objective of this study was to explore the possibility of using CoCl2 to induce mimetic-hypoxia for NPCs and the comparison with hypoxia (1% O2) in vitro. Materials and methods: Rat nucleus pulposus cells of Passage 3-5 were used in this research. Cell viability, rate of cell apoptosis, ROS (reactive oxygen species) generation, cell migration, extracellular pH and extracellular matrix metabolism were determined to compare the influence of hypoxia (1% O2) and CoCl2 on NPCs. Results: We found that the effects of CoCl2 on NPCs was dose-dependent. At the proper concentration, CoCl2 could be used to elicit chemical hypoxia for nucleus pulposus cells in vitro and many biological effects, analogous to physical hypoxia (1% O2), could be achieved such as enhanced cell viability, decreased apoptosis and activated extracellular matrix metabolism. On the other hand, CoCl2 mimetic-hypoxia did not affect NPCs glycolysis and migration compared to physical hypoxia. In addition, high concentration of CoCl2 (>200 µM) is harmful to NPCs with high rates of apoptosis and ECM (extracellular matrix) degradation. Conclusions: It is feasible and convenient to use CoCl2 to induce chemical mimetic hypoxia for culturing NPCs on the premise of appropriate concentration. But in aspects of cell migration and glycolysis, CoCl2 could not achieve similar results with physical hypoxia. This study may provide a convenient method and enlightenment to induce mimetic-hypoxia for researchers studying NPCs and IVVD.

Adjuvant surgical decision-making system for lumbar intervertebral disc herniation after percutaneous endoscopic lumber discectomy: a retrospective nonlinear multiple logistic regression prediction model based on a large sample.

BACKGROUND CONTEXT: Lumbar disc herniation (LDH) is a common condition that can affects an individual' quality of life. In patients for whom conservative treatment is ineffective after 3 months, surgical treatment, such as percutaneous endoscopic lumbar discectomy (PELD), is recommended. Because PELD is minimally invasive and produces thorough nerve root decompression, both surgeons and patients often prefer it to other techniques. PURPOSE: Surgeons find it challenging to prevent postoperative recurrent LDH (rLDH) when they use PELD. We created and verified a model for evaluating patients' recurrence risk factors before surgery so that surgeons can choose other surgical techniques when necessary. STUDY DESIGN: Retrospective study. PATIENT SAMPLE: One thousand eight hundred seven patients who underwent PELD at our hospital between 2012 and 2015 were enrolled. OUTCOME MEASURE: The main outcome measure was rLDH at any follow-up time point. METHODS: Data were retrospectively analyzed for 1807 patients who underwent PELD at our hospital at some point between 2012 and 2015; all patients had been monitored for at least 5 years after surgery. They were divided into a recurrence group and a nonrecurrence group. Clinical and radiological risk factors were assessed over time to determine their correlations with recurrence and to exclude less important factors. A nonlinear multivariate logistic regression model was established to predict the recurrence rate before surgery. RESULTS: A total of 1706 patients were monitored after PELD; data were missing for 101 additional patients. The total recurrence rate was 10.38%, and the most common time from surgery to recurrence was 1 year. Ten risk factors were assessed and included in the analysis. Regarding clinical risk factors, patients with hypertension (p < .001; correlation coefficient R [R] = 0.235; odds ratio [OR] = 4.749), diabetes (p < .001; R = 0.381; OR = 16.797), a history of smoking (p < .001; R = 0.347; OR = 9.012), and a history of performing intense physical labor (p < .001; R = 0.409; OR = 19.592) had a higher recurrence rate. Regarding radiological risk factors, patients with disc degeneration (Pfirrmann grade III) (p < .001; R = 0.228; OR = 4.919), Modic changes (level 2) (p < .001; R = 0.309; OR = 7.934), herniation in the form of extrusion (p < .001; R = 0.365; OR = 12.228), a higher disc height index (DHI) (p < .001; R = 0.336), and a larger segmental range of motion (p < .001; R = 0.243) had a higher recurrence rate. When the lumbar motion angle was negative (p < .001; R = 0.318; OR = 13.680), the recurrence rate was high. The overall accuracy of the final model was 97.6% (1665 of 1706). The recognition rate for non-rLDH cases was 99.0% (1514 of 1529), and the rate for rLDH cases was 85.3% (151 of 177); the AUC was 0.9315. A simple model was used. For those patients with postoperative trauma (p < .001; R = 0.382; OR = 13.680), a comparison model was established, and the corresponding recurrence rate was 23.0% ± 25.0% (0-76%). CONCLUSIONS: A large cohort of patients underwent long-term monitoring, and 11 risk factors were verified for assessing each patient's risks before surgery to predict the postoperative recurrence of LDH following PELD. The risk of recurrence may be effectively reduced with the use of alternative surgical techniques in high risk cases.

Exosomes derived from stem cells as an emerging therapeutic strategy for intervertebral disc degeneration.

Intervertebral disc (IVD) degenerative diseases are a common problem in the world, and they cause substantial social and economic burdens for people. The current methods for treating IVD degenerative diseases mainly include surgery and conservative treatment, which cannot fundamentally restore the normal structure of the disc. With continuous research on the mechanism of degeneration and the development of regenerative medicine, rapid progress has been made in the field of regenerative medicine regarding the use of stem cell-derived exosomes, which are active biological substances used in intercellular communication, because they show a strong effect in promoting tissue regeneration. The study of exosomes in the field of IVD degeneration has just begun, and many surprising achievements have been made. This paper mainly reviews the biological characteristics of exosomes and highlights the current status of exosomes in the field of IVD degeneration, as well as future developments regarding exosomes.

Experimental study of PLLA/INH slow release implant fabricated by three dimensional printing technique and drug release characteristics in vitro.

BACKGROUND: Local slow release implant provided long term and stable drug release in the lesion. The objective of this study was to fabricate biodegradable slow release INH/PLLA tablet via 3 dimensional printing technique (3DP) and to compare the drug release characteristics of three different structured tablets in vitro. METHODS: Three different drug delivery systems (columnar-shaped tablet (CST), doughnut-shaped tablet (DST) and multilayer doughnut-shaped tablet (MDST)) were manufactured by the three dimensional printing machine and isoniazid was loaded into the implant. Dynamic soaking method was used to study the drug release characteristics of the three implants. MTT cytotoxicity test and direct contact test were utilized to study the biocompatibility of the implant. The microstructures of the implants' surfaces were observed with electron microscope. RESULTS: The PLLA powder in the tablet could be excellently combined through 3DP without disintegration. Electron microscope observations showed that INH distributed evenly on the surface of the tablet in a "nest-shaped" way, while the surface of the barrier layer in the multilayer doughnut shaped tablet was compact and did not contain INH. The concentration of INH in all of the three tablets were still higher than the effective bacteriostasis concentration (Isoniazid: 0.025 ~ 0.05 µg/ml) after 30 day's release in vitro. All of the tablets showed initial burst release of the INH in the early period. Drug concentration of MDST became stable and had little fluctuation starting from the 6th day of the release. Drug concentration of DST and CST decreased gradually and the rate of decrease in concentration was faster in DST than CST. MTT cytotoxicity test and direct contact test indicated that the INH-PLLA tablet had low cytotoxicity and favorable biocompatibility. CONCLUSIONS: Three dimensional printing technique was a reliable technique to fabricate complicated implants. Drug release pattern in MDST was the most stable among the three implants. It was an ideal drug delivery system for the antibiotics. Biocompatibility tests demonstrated that the INH-PLLA implant did not have cytotoxicity. The multilayer donut-shaped tablet provided a new constant slow release method after an initial burst for the topical application of the antibiotic.

Bone induction by biomimetic PLGA copolymer loaded with a novel synthetic RADA16-P24 peptide in vivo.

Bone morphogenetic protein-2 (BMP-2) is a key bone morphogenetic protein, and poly(lactic-co-glycolic acid) (PLGA) has been widely used as scaffold for clinical use to carry treatment protein. In the previous studies, we have synthesized BMP-2-related peptide (P24) and found its capacity of inducing bone regeneration. In this research, we have synthesized a new amphiphilic peptide Ac-RADA RADA RADA RADA S[PO4]KIPKASSVPTELSAISTLYLDDD-CONH2 (RADA16-P24) with an assembly peptide RADA16-Ion the P24 item of BMP2 to form divalent ion-induced gelatin. Two methods of physisorption and chemical cross-linking were used to bind RADA16-P24 onto the surface of the copolymer PLGA to synthesize RADA16-P24-PLGA, and its capacity of attaching bone marrow stromal cells (BMSCs) was evaluated in vitro and inducing ectopic bone formation was examined in vivo. In vitro our results demonstrated that RADA16-P24-PLGA copolymer prepared by physisorbing or prepared by chemical cross-linking had a peptide binding rate of (2.0180±0.5296)% or (10.0820±0.8405)% respectively (P<0.05). In addition the BMSCs proliferated vigorously in the RADA16-P24-PLGA biomaterials. Significantly the percentage of BMSCs attached to RADA16-P24-PLGA composite prepared by chemical cross-linking and physisorbing were (71.4±7.5) % or (46.7±5.8) % (P<0.05). The in vivo study showed that RADA16-P24-PLGA chemical cross-linking could better induce ectopic bone formation compared with RADA16-P24-PLGA physisorbing and PLGA. It is concluded that the PLGA copolymer is a good RADA16-P24 carrier. This novel RADA16-P24-PLGA composite has strong osteogenic capability.