Vitamin C Deficiency Deteriorates Bone Microarchitecture and Mineralization in a Sex-Specific Manner in Adult Mice.

Vitamin C (VitC) is essential for bone health, and low VitC serum levels increase the risk for skeletal fractures. If and how VitC affects bone mineralization is unclear. Using micro-computed tomography (µCT), histologic staining, as well as quantitative backscattered electron imaging (qBEI), we assessed the effects of VitC on femoral structure and microarchitecture, bone formation, and bone mineralization density distribution (BMDD) in the VitC incompetent Gulo-/- mouse model and wild-type mice. In particular, VitC-supplemented, 20-week-old mice were compared with age-matched counterparts where dietary VitC intake was excluded from week 15. VitC depletion in Gulo-/- mice severely reduced cortical thickness of the diaphyseal shaft and bone volume around the growth plate (eg, bone volume of the primary spongiosa -43%, p < 0.001). Loss of VitC also diminished the amount of newly formed bone tissue as visualized by histology and calcein labeling of the active mineralization front. BMDD analysis revealed a shift to higher calcium concentrations upon VitC supplementation, including higher average (~10% increase in female VitC deficient mice, p < 0.001) and peak calcium concentrations in the epiphyseal and metaphyseal spongiosa. These findings suggest higher bone tissue age. Importantly, loss of VitC had significantly more pronounced effects in female mice, indicating a higher sensitivity of their skeleton to VitC deficiency. Our results reveal that VitC plays a key role in bone formation rate, which directly affects mineralization. We propose that low VitC levels may contribute to the higher prevalence of bone-degenerative diseases in females and suggest leveraging this vitamin against these conditions. © 2023 American Society for Bone and Mineral Research (ASBMR).

Polysaccharide from Angelica sinensis attenuates SNP-induced apoptosis in osteoarthritis chondrocytes by inducing autophagy via the ERK1/2 pathway.

OBJECTIVE: Chondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism. METHOD: Human primary chondrocytes isolated from the articular cartilage of OA patients were treated with SNP alone or in combination with different doses of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3) II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localization, and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile, activation of the ERK 1/2 pathway was determined by western blot. The autophagy inhibitors, 3-methyladenine (3-MA), chloroquine (CQ), and a specific inhibitor of ERK1/2, SCH772984, were used to confirm the autophagic effect of ASP. RESULTS: The results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocyte proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of the ERK1/2 pathway. CONCLUSION: ASP markedly rescued SNP-induced apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP as a potential therapeutic supplementation for OA treatment.

Reduction of arthrofibrosis utilizing a collagen membrane drug-eluting scaffold with celecoxib and subcutaneous injections with ketotifen.

The dense formation of abnormal scar tissue after total knee arthroplasty results in arthrofibrosis, an unfortunate sequela of inflammation. The purpose of this study was to use a validated rabbit model to assess the effects on surgically-induced knee joint contractures of two combined pharmacological interventions: celecoxib (CXB) loaded on an implanted collagen membrane, and subcutaneously (SQ) injected ketotifen. Thirty rabbits were randomly divided into five groups. The first group received no intervention after the index surgery. The remaining four groups underwent intra-articular implantation of collagen membranes loaded with or without CXB at the time of the index surgery; two of which were also treated with SQ ketotifen. Biomechanical joint contracture data were collected at 8, 10, 16, and 24 weeks. At the time of necropsy (24 weeks), posterior capsule tissue was collected for messenger RNA and histopathologic analyses. At 24 weeks, there was a statistically significant increase in passive extension among rabbits in all groups treated with CXB and/or ketotifen compared to those in the contracture control group. There was a statistically significant decrease in COL3A1, COL6A1, and ACTA2 gene expression in the treatment groups compared to the contracture control group (P < .001). Histopathologic data also demonstrated a trend towards decreased fibrous tissue density in the CXB membrane group compared to the vehicle membrane group. The present data suggest that intra-articular placement of a treated collagen membrane blunts the severity of contracture development in a rabbit model of arthrofibrosis, and that ketotifen and CXB may independently contribute to the prevention of arthrofibrosis. Statement of clinical significance: Current literature has demonstrated that arthrofibrosis may affect up to 5% of primary total knee arthroplasty patients. For that reason, novel pharmacologic prophylaxis and treatment modalities are critical to mitigating reoperations and revisions while improving the quality of life for patients with this debilitating condition.

Joint contracture is reduced by intra-articular implantation of rosiglitazone-loaded hydrogels in a rabbit model of arthrofibrosis.

Trauma, surgery, and other inflammatory conditions can lead to debilitating joint contractures. Adjunct pharmacologic modalities may permit clinical prevention and treatment of recalcitrant joint contractures. We investigated the therapeutic potential of rosiglitazone by intra-articular delivery via oligo[poly(ethylene glycol)fumarate] (OPF) hydrogels in an established rabbit model of arthrofibrosis. OPF hydrogels loaded with rosiglitazone were characterized for drug elution properties upon soaking in minimum essential media (MEM) with 10% fetal bovine serum and measurements of drug concentrations via High Performance Liquid Chromatography (HPLC). Drug-loaded scaffolds were surgically implanted into 24 skeletally mature female New Zealand White rabbits that were divided into equal groups receiving OPF hydrogels loaded with rosiglitazone (1.67 mg), or vehicle control (10 µl DMSO). After 8 weeks of joint immobilization, rabbits were allowed unrestricted cage activity for 16 weeks. Contracture angles of rabbit limbs treated with rosiglitazone showed statistically significant improvements in flexion compared to control animals (mean angles, respectively, 64.4° vs. 53.3°, p < 0.03). At time of sacrifice (week 24), animals in the rosiglitazone group continued to exhibit less joint contracture than controls (119.0° vs. 99.5°, p = 0.014). The intra-articular delivery of rosiglitazone using implanted OPF hydrogels decreases flexion contractures in a rabbit model of arthrofibrosis without causing adverse effects (e.g., gross inflammation or arthritis). Statement of Clinical Significance: Post-traumatic joint contractures are common and debilitating, with limited available treatment options. Pharmacologic interventions can potentially prevent and treat such contractures. This study is translational in that a commercially approved medication has been repurposed through a novel delivery device. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2949-2955, 2018.

Cytotoxicity of Local Anesthetics in Mesenchymal Stem Cells.

Cell therapy based on the trophic, mitogenic, and immunomodulatory capacity of mesenchymal stem cells is a promising treatment modality for degenerative musculoskeletal conditions. Local anesthetics have been commonly used in interventional procedures for alleviating pain, but local anesthetics may have negative impact on MSC dosing because of cytotoxicity or other biological effects. Because previous studies have not reached consensus yet on the potential complications of local anesthetics in cell therapy, we reviewed 11 studies that involve in vitro experimentation with MSCs using aminoamide-type anesthetics including lidocaine, ropivacaine, mepivacaine, bupivacaine, articaine, and prilocaine. Three studies that compare the effects of different types of local anesthetic agents showed that ropivacaine has the least detrimental effects on mesenchymal stem cell populations, whereas lidocaine seems to have the most significant effects on stem cell viability. Concentration- and time-dependent effects on cell viability were reported with bupivacaine, ropivacaine, lidocaine, and mepivacaine. We conclude that local anesthetic agents have time- and concentration-dependent detrimental effects on MSCs. However, in vivo studies will be required to understand the interactions of these agents with MSCs, because in vitro studies cannot replicate the pharmacokinetics of anesthetics in vivo or the recovery of MSCs in a more physiological environment.

Ethanol Extract of Cissus quadrangularis Enhances Osteoblast Differentiation and Mineralization of Murine Pre-Osteoblastic MC3T3-E1 Cells.

Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti-osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre-osteoblast cell line, MC3T3-E1. Ethanolic extract of CQ (CQ-E) was found to affect growth kinetics of MC3T3-E1 cells in a dosage-dependent manner. High concentrations of CQ-E (more than 10 µg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non-toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ-E. CQ-E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose-dependent effect of CQ-E with lower concentrations exhibiting anabolic and osteogenic properties. J. Cell. Physiol. 232: 540-547, 2017. © 2016 Wiley Periodicals, Inc.

A conserved interaction that is essential for the biogenesis of histone locus bodies.

Nuclear protein, ataxia-telangiectasia locus (NPAT) and FLICE-associated huge protein (FLASH) are two major components of discrete nuclear structures called histone locus bodies (HLBs). NPAT is a key co-activator of histone gene transcription, whereas FLASH through its N-terminal region functions in 3' end processing of histone primary transcripts. The C-terminal region of FLASH contains a highly conserved domain that is also present at the end of Yin Yang 1-associated protein-related protein (YARP) and its Drosophila homologue, Mute, previously shown to localize to HLBs in Drosophila cells. Here, we show that the C-terminal domain of human FLASH and YARP interacts with the C-terminal region of NPAT and that this interaction is essential and sufficient to drive FLASH and YARP to HLBs in HeLa cells. Strikingly, only the last 16 amino acids of NPAT are sufficient for the interaction. We also show that the C-terminal domain of Mute interacts with a short region at the end of the Drosophila NPAT orthologue, multi sex combs (Mxc). Altogether, our data indicate that the conserved C-terminal domain shared by FLASH, YARP, and Mute recognizes the C-terminal sequence of NPAT orthologues, thus acting as a signal targeting proteins to HLBs. Finally, we demonstrate that the C-terminal domain of human FLASH can be directly joined with its N-terminal region through alternative splicing. The resulting 190-amino acid MiniFLASH, despite lacking 90% of full-length FLASH, contains all regions necessary for 3' end processing of histone pre-mRNA in vitro and accumulates in HLBs.

The rat intervertebral disk degeneration pain model: relationships between biological and structural alterations and pain.

INTRODUCTION: Degeneration of the interverterbral disk is as a cause of low-back pain is increasing. To gain insight into relationships between biological processes, structural alterations and behavioral pain, we created an animal model in rats. METHODS: Disk degeneration was induced by removal of the nucleus pulposus (NP) from the lumbar disks (L4/L5 and L5/L6) of Sprague Dawley rats using a 0.5-mm-diameter microsurgical drill. The degree of primary hyperalgesia was assessed by using an algometer to measure pain upon external pressure on injured lumbar disks. Biochemical and histological assessments and radiographs of injured disks were used for evaluation. We investigated therapeutic modulation of chronic pain by administering pharmaceutical drugs in this animal model. RESULTS: After removal of the NP, pressure hyperalgesia developed over the lower back. Nine weeks after surgery we observed damaged or degenerated disks with proteoglycan loss and narrowing of disk height. These biological and structural changes in disks were closely related to the sustained pain hyperalgesia. A high dose of morphine (6.7 mg/kg) resulted in effective pain relief. However, high doses of pregabalin (20 mg/kg), a drug that has been used for treatment of chronic neuropathic pain, as well as the anti-inflammatory drugs celecoxib (50 mg/kg; a selective inhibitor of cyclooxygenase 2 (COX-2)) and ketorolac (20 mg/kg; an inhibitor of COX-1 and COX-2), did not have significant antihyperalgesic effects in our disk injury animal model. CONCLUSIONS: Although similarities in gene expression profiles suggest potential overlap in chronic pain pathways linked to disk injury or neuropathy, drug-testing results suggest that pain pathways linked to these two chronic pain conditions are mechanistically distinct. Our findings provide a foundation for future research on new therapeutic interventions that can lead to improvements in the treatment of patients with back pain due to disk degeneration.

Identification of HiNF-P, a key activator of cell cycle-controlled histone H4 genes at the onset of S phase.

At the G(1)/S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G(1)/S phase transition.

The tumor suppressor interferon regulatory factor 1 interferes with SP1 activation to repress the human CDK2 promoter.

Cell growth control by interferons (IFNs) involves up-regulation of the tumor suppressor interferon regulatory factor 1 (IRF1). To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here we show that the G1/S phase-related cyclin-dependent kinase 2 (CDK2) gene is a novel proliferation-related downstream target of IRF1. We find that IRF1, but not IRF2, IRF3, or IRF7, selectively represses CDK2 gene transcription in a dose- and time-dependent manner. We delineate the IRF1-responsive repressor element between nt -68 to -31 of the CDK2 promoter. For comparison, the tumor suppressor p53 represses CDK2 promoter activity independently of IRF1 through sequences upstream of nt -68, and the CDP/cut/Cux1 homeodomain protein represses transcription down-stream of -31. Thus, IRF1 repression represents one of three distinct mechanisms to attenuate CDK2 levels. The -68/-31 segment lacks a canonical IRF responsive element but contains a single SP1 binding site. Mutation of this element abrogates SP1-dependent enhancement of CDK2 promoter activity as expected but also abolishes IRF1-mediated repression. Forced elevation of SP1 levels increases endogenous CDK2 levels, whereas IRF1 reduces both endogenous SP1 and CDK2 protein levels. Hence, IRF1 represses CDK2 gene expression by interfering with SP1-dependent transcriptional activation. Our findings establish a causal series of events that functionally connect the anti-proliferative effects of interferons with the IRF1-dependent suppression of the CDK2 gene, which encodes a key regulator of the G1/S phase transition.