HuThe purpose of this form is to provide readers of your manuscript with information about your other interests that could influence how they receive and understand your work. The form is designed to be completed electronically and stored electronically. It contains programming that allows appropriate data display. Each author should submit a separate form and is responsible for the accuracy and completeness of the submitted information. The form is in six parts.

Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.

No abstract

Acute spinal cord injury (SCI) induces secondary hemorrhage and initial blood-spinal cord barrier (BSCB) disruption. The transient receptor potential melastatin 4 (Trpm4) together with sulfonylurea receptor 1 (Sur1) forms the Sur1-Trpm4 channel complex. The up-regulation of Sur1-Trpm4 after injury plays a crucial role in secondary hemorrhage, which is the most destructive mechanism in secondary injuries of the central nervous system (CNS). The matrix metalloprotease (MMP)-mediated disruption of the BSCB leads to an inflammatory response, neurotoxin production and neuronal cell apoptosis. Thus, preventing secondary hemorrhage and BSCB disruption should be an important goal of therapeutic interventions in SCI.Methods: Using a moderate contusion injury model at T10 of the spinal cord, flufenamic acid (FFA) was injected intraperitoneally 1 h after SCI and then continuously once per day for one week.Results: Trpm4 expression is highly up-regulated in capillaries 1 d after SCI. Treatment with flufenamic acid (FFA) inhibited Trpm4 expression, secondary hemorrhage, and capillary fragmentation and promoted angiogenesis. In addition, FFA significantly inhibited the expression of MMP-2 and MMP-9 at 1 d after SCI and significantly attenuated BSCB disruption at 1 d and 3 d after injury. Furthermore, we found that FFA decreased the hemorrhage- and BSCB disruption-induced activation of microglia/macrophages and was associated with smaller lesions, decreased cavity formation, better myelin preservation and less reactive gliosis. Finally, FFA protected motor neurons and improved locomotor functions after SCI.Conclusion: This study indicates that FFA improves functional recovery, in part, due to the following reasons: (1) it inhibits the expression of Trpm4 to reduce the secondary hemorrhage; and (2) it inhibits the expression of MMP-2 and MMP-9 to block BSCB disruption. Thus, the results of our study suggest that FFA may represent a potential therapeutic agent for promoting functional recovery.

The ideal bone graft material must be biocompatible, biodegradable, osteoconductive and osteoinductive. In this study, a new biomimetic scaffold based on mineralized recombinant collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA), was prepared and the synthetic P24 peptide derived from BMP-2 was introduced into the porous nHA/RHLC/PLA scaffold to improve its osteoinductive property. The nHA/RHLC/PLA implants loaded with 3 mg, 2 mg, 1 mg and 0 mg P24 peptide were implanted subcutaneously into rats. At the 4th, 8th and 12th weeks after implantation, the rats were sacrificed in batch and the samples were harvested. Their osteogenic capability was detected by CT scan and histological observation. The results indicated that the osteogenic capability of 3 mg, 2 mg and 1 mg of the P24 peptide was superior to the implants without the P24 peptide. There was no significant difference between implants with 3 mg and 2 mg P24 peptide, but the osteogenic capability of the two dosage groups was significantly better than that of the 1 mg group. It was concluded that BMP-2-derived peptide can increase the osteoinduction of nHA/RHLC/PLA scaffold and the P24 peptide induced new bone formation in a dose-dependent manner. The nHA/RHLC/PLA scaffold loaded with the synthetic BMP-2-derived peptide is a kind of ideal scaffold material for bone tissue engineering.

Intervertebral disc (IVD) degeneration (IVDD) is the main cause of low back pain with major social and economic burdens; however, its underlying molecular mechanisms remain poorly defined. Here we show that the focal adhesion protein Kindlin-2 is highly expressed in the nucleus pulposus (NP), but not in the anulus fibrosus and the cartilaginous endplates, in the IVD tissues. Expression of Kindlin-2 is drastically decreased in NP cells in aged mice and severe IVDD patients. Inducible deletion of Kindlin-2 in NP cells in adult mice causes spontaneous and striking IVDD-like phenotypes in lumbar IVDs and largely accelerates progression of coccygeal IVDD in the presence of abnormal mechanical stress. Kindlin-2 loss activates Nlrp3 inflammasome and stimulates expression of IL-1β in NP cells, which in turn downregulates Kindlin-2. This vicious cycle promotes extracellular matrix (ECM) catabolism and NP cell apoptosis. Furthermore, abnormal mechanical stress reduces expression of Kindlin-2, which exacerbates Nlrp3 inflammasome activation, cell apoptosis, and ECM catabolism in NP cells caused by Kindlin-2 deficiency. In vivo blocking Nlrp3 inflammasome activation prevents IVDD progression induced by Kindlin-2 loss and abnormal mechanical stress. Of translational significance, adeno-associated virus-mediated overexpression of Kindlin-2 inhibits ECM catabolism and cell apoptosis in primary human NP cells in vitro and alleviates coccygeal IVDD progression caused by mechanical stress in rat. Collectively, we establish critical roles of Kindlin-2 in inhibiting Nlrp3 inflammasome activation and maintaining integrity of the IVD homeostasis and define a novel target for the prevention and treatment of IVDD.

Excessive compression, the main cause of intervertebral disc (IVD) degeneration, affected endogenous repair of the intervertebral disc. Pioglitazone (PGZ) is the agonist of peroxisome proliferator-activated receptor γ, which has been widely used in the treatment of diabetes mellitus. The present study aim at investigating whether pioglitazone has protective effects on compression-mediated cell apoptosis in nucleus pulposus mesenchymal stem cells (NP-MSCs) and further exploring the possible underlying mechanism. Our results indicated that the isolated cells satisfied the criteria of MSC stated by the International Society for Cellular Therapy. Besides, our research revealed that pioglitazone could protect cell viability, cell proliferation of NP-MSCs and alleviated the toxic effects caused by compression. The actin stress fibers was suppressed obviously under compression, and pioglitazone alleviated the adverse outcomes. Pioglitazone exerted protective effects on compression-induced NP-MSCs apoptosis according to annexin V/PI double-staining and TUNEL assays. Pioglitazone suppressed compression-induced NP-MSCs oxidative stress, including decreasing compression-induced overproduction of reactive oxygen species (ROS) and malondialdehyde (MDA), and alleviated compression-induced mitochondrial membrane potential (MMP) decrease. Ultrastructure collapse of the mitochondria exhibited a notable improvement by pioglitazone in compression-induced NP-MSCs according to transmission electron microscopy (TEM). Furthermore, the molecular results showed that pioglitazone significantly decreased the expression of apoptosis-associated proteins, including cyto.cytochrome c, Bax, cleaved caspase-9, and cleaved caspase-3, and promoted Bcl-2 expression. These results indicated that pioglitazone alleviated compression-induced NP-MSCs apoptosis by suppressing oxidative stress and the mitochondrial apoptosis pathway, which may be a valuable candidate for the treatment of IVD degeneration.

In this study, a new functionalized peptide RLN was designed containing the bioactive motif link N, the amino terminal peptide of link protein. A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16. The characterization of LN-NS was tested using atomic force microscopy (AFM). The biocompatibility and bioactivity of this nanofiber scaffold for rabbit nucleus pulposus cells (NPCs) were also evaluated. This designer functionalized nanofiber scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously. In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS-guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs. Our findings demonstrate that the functionalized nanofiber scaffold containing link N had excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration.