Nutritional deficiency induces nucleus pulposus cell apoptosis via the ATF4-PKM2-AKT signal axis.

BACKGROUND: The intervertebral disc is the largest avascular tissue in the human body. The nucleus pulposus (NP) consumes glucose and oxygen to generate energy to maintain cellular metabolism via nutrients that diffuse from the cartilage endplate. The microenvironment in the intervertebral disc becomes nutritionally deficient during degeneration, and nutritional deficiency has been shown to inhibit the viability and proliferation of NP cells. METHODS: To investigate the molecular mechanism by which nutritional deficiency reduces viability and decreases proliferation, we created an in vitro model by using decreasing serum concentration percentages. RESULTS: In this study, we found that nutritional deficiency reduced NP cell viability and increased cell apoptosis and that the upregulation of ATF4 expression and the downregulation of PKM2 expression were involved in this process. Moreover, we found that PKM2 inhibition can reduce the cell apoptosis induced by ATF4 silence under nutritional deficiency. CONCLUSION: Our findings revealed that PKM2 inhibition reduces the cell apoptosis induced by ATF4 silence under nutritional deficiency by inhibiting AKT phosphate. Revealing the function and mechanism of NP cell development under nutritional deficiency will provide new insights into the etiology, diagnosis, and treatment of intervertebral disc and related diseases.

Combined Effects of Single Nucleotide Polymorphisms (SNPs) within C-reactive Protein (CRP) and Environmental Parameters on Risk and Prognosis for Diabetic Foot Osteomyelitis Patients.

PURPOSE: This study was aimed to discover the combined effects of single nucleotide polymorphisms (SNPs) within the C-reactive protein (CRP) gene and potential environmental factors on the risk and prognosis for diabetic foot osteomyelitis (DFO). METHODS: A total of 1734 diabetes mellitus patients, 681 with DFO and 1053 without DFO, were successfully recruited, as well as 1261 healthy control individuals. Participants data were recorded regarding age, gender, smoking and drinking history, body mass index (BMI), hypertension, cacosmia, and ulceration. A total of 11 SNPs within the CRP gene were designated for exploration, by logistic regression analyses, of how they might interact with environmental factors to affect susceptibility to DFO. RESULTS: Frequencies of smoking and drinking, and incidence of hypertension, cacosmia, or ulceration displayed marked differences (all P<0.05) between DFO and non-DFO patients. Furthermore, allele G of rs11265260 (A>G), allele G of rs1800947 (C>G), and allele T of rs3093059 (T>C) and rs1130864 (C>T) exhibited a trend to increase risk of DFO (all P<0.05). Allele G of rs11265260 (A>G), allele G of rs1800947 (C>G) and rs3093068 (G>C), and allele T of rs1130864 (C>T) were significant predictors of poor prognosis among DFO patients (P<0.05). In addition, genotypes of rs11265260 (i.e., GG and AG), rs1800947 (i.e., CG and GG), rs3093059 (i.e., TT) and rs113084 (i.e., CT and TT) amplified the influence of smoking, alcohol consumption, cacosmia, and ulceration on progression from non-DFO to DFO (all γ>1). CONCLUSION: Genetic mutations within CRP functioned interactively with external factors to affect DFO risk.

LncRNA MEG3 negatively modified osteosarcoma development through regulation of miR-361-5p and FoxM1.

This study was aimed to figure out whether long noncoding RNA MEG3/miR-361-5p/FoxM1 signaling would contribute to improved proliferation and metastasis of osteosarcoma cells. We altogether collected 204 pairs of osteosarcoma tissues and adjacent normal tissues, and obtained four human osteosarcoma cell lines. Then pcDNA3.1-MEG3, si-MEG3, miR-361-5p mimic, miR-361-5p inhibitor, pcDNA3.1-FoxM1, si-FoxM1, and negative control (NC) were, respectively, transfected into the osteosarcoma cells. Furthermore, real time polymerase chain reaction was utilized to determine the mRNA expressions of maternally expressed gene 3 (MEG3) and miR-361-5p, and western blot analysis was applied for determining the FoxM1 expression. Besides, dual luciferase reporter gene assay was adopted to verify if MEG3 can be directly targeted by miR-361-5p. Finally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, colony formation assay, flow cytometry, wound healing assay, and transwell assay were conducted to investigate the influence of MEG3, miR-361-5p, and FoxM1 expressions on the viability, proliferation, apoptosis, migration, and invasion of osteosarcoma cells. MEG3 and miR-361-5p were observed to be significantly downregulated within both osteosarcoma tissues and cell lines, whereas FoxM1 was upregulated in osteosarcoma tissues and cell lines (p < 0.05). MEG3 directly bound to miR-361-5p, and significantly upgraded its expression (p < 0.05). The upregulated MEG3 and miR-361-5p or the downregulated FoxM1 appeared to substantially inhibit proliferation, migration, and invasion of osteosarcoma cells (p < 0.05). Finally, the proliferation, migration, invasion, and motility of osteosarcoma cells within the miR-NC + pcDNA3.1-FoxM1 group and pcDNA + pcDNA-FoxM1 group were markedly promoted when compared with the miR-361-5p mimic group and pcDNA3.1-MEG3 group (p < 0.05). The MEG3/miR-361-5p/FoxM1 axis could potentially serve as therapeutic targets or diagnostic biomarkers for osteosarcoma.

Preparing Platelet-Rich Plasma with Whole Blood Harvested Intraoperatively During Spinal Fusion.

BACKGROUND Platelet-rich plasma (PRP) has gained growing popularity in use in spinal fusion procedures in the last decade. Substantial intraoperative blood loss is frequently accompanied with spinal fusion, and it is unknown whether blood harvested intraoperatively qualifies for PRP preparation. MATERIAL AND METHODS Whole blood was harvested intraoperatively and venous blood was collected by venipuncture. Then, we investigated the platelet concentrations in whole blood and PRP, the concentration of growth factors in PRP, and the effects of PRP on the proliferation and viability of human bone marrow-derived mesenchymal stem cells (HBMSCs). RESULTS Our results revealed that intraoperatively harvested whole blood and whole blood collected by venipuncture were similar in platelet concentration. In addition, PRP formulations prepared from both kinds of whole blood were similar in concentration of platelet and growth factors. Additional analysis showed that the similar concentrations of growth factors resulted from the similar platelet concentrations of whole blood and PRP between the two groups. Moreover, these two kinds of PRP formulations had similar effects on promoting cell proliferation and enhancing cell viability. CONCLUSIONS Therefore, intraoperatively harvested whole blood may be a potential option for preparing PRP spinal fusion.

Nano-carrier mediated co-delivery of methyl prednisolone and minocycline for improved post-traumatic spinal cord injury conditions in rats.

OBJECTIVE: The objective of this study is to investigate the fate of albumin coupled nanoparticulate system over non-targeted drug carrier in the treatment of hemisectioned spinal cord injury (SCI). SIGNIFICANCE: Targeted delivery of methyl prednisolone (MP) and minocycline (MC) portrayed improved therapeutic efficacy as compared with non-targeted nanoparticles (NPS). METHODS: Albumin coupled, chitosan stabilized, and cationic NPS (albumin-MP + MC - NPS) of poly-(lactide-co-glycolic acid) were prepared using the emulsion solvent evaporation method. Prepared NPS were characterized for drug entrapment efficiency, particle size, poly-dispersity index (PDI), zeta potential, and morphological characteristics. Their evaluation was done based on the pharmaceutical, toxicological, and pharmacological parameters. RESULTS AND DISCUSSION: In vitro release of MP + MC from albumin-MP + MC - NPS and MP + MC - NPS was observed to be very controlled for the period of eight days. Cell viability study portrayed non-toxic nature of the developed NPS. Albumin-MP + MC - NPS showed prominent anti-inflammatory potential as compared with non-targeted NPS (MP + MC - NPS) when studied in LPS-induced inflamed astrocytes. Albumin-MP + MC - NPS reduced lesional volume and improved behavioral outcomes significantly in rats with SCI (hemisectioned injury model) when compared with that of MP + MC - NPS. CONCLUSIONS: Albumin-coupled NPS carrier offered an effective method of SCI treatment following safe co-administration of MP and MC. The in vitro and in vivo effectiveness of MP + MC was improved tremendously when compared with the effectiveness showed by MP + MC - NPS. That could be attributed to the site specific, controlled release of MP + MC to the inflammatory site.

Effect of a bioabsorbable, super-high molecular weight poly-D,L-lactic acid plate containing recombinant human bone morphogenetic protein-2 for fracture healing.

The aim of this study was to investigate the effect of a bioabsorbable, super-high molecular weight poly-D,L-lactic acid (PDLLA) plate exhibiting the sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) (PDLLA-rhBMP-2) on the treatment of fracture with internal fixation. A total of 32 New Zealand rabbits were randomly allocated to one of four groups (2, 4, 8 and 12 weeks), and a 2.5-mm middle ulnar osteotomy was performed bilaterally. The right side (experimental side) was fixed internally with PDLLA-rhBMP-2, and the left side (control side) was fixed with a normal PDLLA plate. At 2, 4, 8 and 12 weeks after surgery, the gross pathology of the ulnas was examined and radiographic, histological and computer image analyses were performed. The results demonstrated that the ulna fractures were fixed stably with the two bioactive plates at 2, 4, 8 and 12 weeks after surgery. At the 8-week time-point, 7 rabbits exhibited good healing at the osteotomy site on the experimental side. At 12 weeks after surgery, 8 rabbits exhibited good healing at the osteotomy site on both sides, but the experimental side showed enhanced compatibility between the plates and surrounding tissue, faster bone formation, a greater bone regeneration mass and better medullary canal structure compared with the control side. In conclusion, PPLLA-rhBMP-2 may be effectively used to treat fracture or nonunion at a non-weight-bearing site.

[Clinical application of bioactive CPC loading rhBMP-2 in repairing bone defects].

OBJECTIVE: To investigate the clinical application of self-setting CPC loading rhBMP-2 for repair of bone defects and to evaluate the clinical effect and safety. METHODS: From June 2006 to September 2007, 112 bone defects patients were treated by CPC loading rhBMP-2 (rhBMP-2/CPC group) or CPC (control group). The range of bone defect was from 1 cm x 1 cm x 1 cm to 4 cm x 3 cm x 3 cm. In the control group, 63 patients included 31 males and 32 females, aging from 17 to 70 years with an average of 47.4 years. The bone defects were located as follows: calcaneus in 19 patients, tibial plateau in 20 patients, proximal humerus in 8 patients, distal radius in 9 patients and thoracolumbar vertebrae in 7 patients. In the rhBMP-2/CPC group, 49 patients included 31 males and 18 females, aging from 16 to 68 years with an average of 45.6 years. The bone defects were located as follows: calcaneus in 11 patients, tibial plateau in 16 patients, proximal humerus in 7 patients, distal radius in 2 patients, distal tibia in 2 patients and thoracolumbar vertebrae in 11 patients. All defects were repaired with rhBMP-2/CPC (2-5 g) and CPC (2-50 g) in the rhBMP-2/CPC group and the control group, respectively. RESULTS: A total of 108 patients got primary healing after operation. Incisions oozing light yellow fluids were found in 4 patients (control group in 1, rhBMP-2/CPC group in 3), and then healed through dressing changes and taking glucocorticoid. There were no allergic or toxic reaction, no rush or high fever, no fluctuation of hepatic and renal function, blood routine, CRP and urine routine. All patients were followed up for 12 to 24 months (mean 13.2 months). The X-ray examination showed that the implanted material was firmly bonded to the bone at the interface and the anatomic contour of the bone at the sites of defects was successfully restored, and no ablation occurred. All patients got bone union after 3 months of operation. The movement and function of flexion and extension of affected limbs recovered to the normal level. CONCLUSION: Repairing bone defects with rhBMP-2/CPC is safe and effective. Using rhBMP-2/CPC is a promising therapy to deal with bone defects.