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Study design:An in vivo study in mouse models of spinal cord contusion.Objectives:To develop a novel indicator to anticipate the severity of spinal cord injury (SCI) during the acute phase and for the assessment of the efficacy of novel therapies. MicroRNAs (miRNAs) circulating in the peripheral blood are reported to modulate signaling between cells, and to be diagnostic markers for cancers. The purpose of this study was to identify circulating miRNAs for predicting the severity of SCI in the acute phase.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.Methods:Mouse SCI models were made using Infinite Horizon impactor with 50 or 70 kdyn compressing power following thoracic laminectomy. The mice were then divided into four groups: normal (without surgery), sham (laminectomy only), mild (50 kdyn), and severe (70 kdyn). TaqMan low-density array analysis and real-time PCR were performed to identify candidate miRNAs that were increased in the serum relative to the severity of SCI.Results:The expression levels of miR-9*, miR-219 and miR-384-5p in the serum were significantly increased relative to the severity of SCI 12 h after injury. The expression of miR-9* was also significantly increased relative to injury severity at 3 and 24 h after injury.Conclusion:Serum miR-9*, miR-219 and miR-384-5p might be promising biomarkers for predicting the severity of SCI. [ABSTRACT FROM AUTHOR]

Study design:We investigated microRNA (miRNA) expression after spinal cord injury (SCI) in mice.Objectives:The recent discovery of miRNAs suggests a novel regulatory control over gene expression during plant and animal development. MiRNAs are short noncoding RNAs that suppress the translation of target genes by binding to their mRNAs, and play a central role in gene regulation in health and disease. The purpose of this study was to examine miRNA expression after SCI.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University.Methods:We examined the expression of miRNA (miR)-223 and miR-124a in a mouse model at 6 h, 12 h, 1 day, 3 days and 7 days after SCI using quantitative PCR. The miRNA expression was confirmed by in situ hybridization.Results:Quantitative PCR revealed two peaks of miR-223 expression at 6 and 12 h and 3 days after SCI. MiR-124a expression decreased significantly from 1 day to 7 days after SCI. In situ hybridization demonstrated the presence of miR-223 around the injured site. However, miR-124a, which was present in the normal spinal cord, was not observed at the injured site.Conclusion:Our results indicate a time-dependent expression pattern of miR-223 and miR-124a in a mouse model of SCI. In this study, the time course of miRNA-223 expression may be related to inflammatory responses after SCI, and the time course of decreased miR-124a expression may reflect cell death. [ABSTRACT FROM AUTHOR]

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs. [ABSTRACT FROM AUTHOR]

The effect of hydrostatic pressure on the viability of soil microorganisms has been examined to develop a new method of sterilization without using agricultural chemicals and to explore the habitable zone of the microorganisms in the earth's interior. It is found that almost all microorganisms in soil cannot survive above 0.75 GPa at room temperature but a small amount of microorganisms are still alive above 0.75 GPa. The slow rate of releasing pressure is found to have a small effect on the viability. [ABSTRACT FROM AUTHOR]

Heterotopic ossification (HO) in Achilles tendon often arises due to endochondral ossification during the healing process following trauma. Retinoic acid receptor γ (RARγ) plays a critical role in this phenomenon. This study aims to elucidate the therapeutic effects of CD1530, an RARγ selective agonist, along with the contributing cells, in Achilles tendon healing, utilizing a cell lineage tracing system. Local injection of CD1530 facilitated histological tendon healing by inhibiting chondrification in a mouse Achilles rupture model. Resident Scleraxis (Scx) + cells in Achilles tendon were not found to be actively involved in HO or　tendon healing following injury. Instead, these processes were primarily driven by tendon stem/progenitor cells (TSPC)-like cells. Furthermore, an in vitro assay revealed that CD1530 attenuated inflammation in injured Achilles tendon-derived tendon fibroblasts (iATF) and inhibited the chondrogenesis of iATF. This dual effect suggests the potential of CD1530 in effectively modulating the healing environment during tendon healing. Together, the present study demonstrated that the local administration of CD1530 accelerated tendon healing by modulating the healing environment, including reducing chondrification via targeting TSPC-like cells in a mouse Achilles tendon rupture model. These results suggest that CD1530 may have the potential to be a novel tendon therapy that offers benefits via the inhibition of chondrogenesis., (© 2024 Orthopaedic Research Society.)

The main pathogenesis of the frozen shoulder is thought to be the inflammation of the intra-articular synovium and subsequent fibrosis of the shoulder joint capsule. However, the molecular pathogenesis of the frozen shoulder is still unknown. A class of noncoding RNAs, microRNAs contribute to various diseases including musculoskeletal diseases. MicroRNA-26a (miR-26a) has been reported to be associated with fibrosis in several organs. This study aims to reveal the role of miR-26a on fibrosis in the shoulder capsule using a frozen shoulder model in miR-26a deficient (miR-26a KO) mice. MiR-26a KO and wild-type (WT) mice were investigated using a frozen shoulder model. The range of motion (ROM) of the shoulder, histopathological changes such as synovitis, and fibrosis-related gene expression in the model mice were evaluated to determine the role of miR-26a. In WT mice, both inflammatory cell infiltration and thickening of the inferior shoulder joint capsule were observed after 1 week of immobilization, and this thickening further progressed over the subsequent 6 weeks. However, the immobilized shoulder in miR-26a KO mice consistently exhibited significantly better ROM compared with WT mice at 1 and 6 weeks, and histological changes were significantly less severe. The expression of inflammation- and fibrosis-related genes was decreased in the miR-26a KO mice compared with WT mice at 1 and 6 weeks. Together, miR-26a deficiency attenuated the severity of frozen shoulder in the immobilization model mouse. The present study suggests that miR-26a has the potential to be a target miRNA for therapeutic approach to frozen shoulder., (© 2024 Orthopaedic Research Society.)

Extracellular vesicles (EVs) have excellent biocompatibility and long retention times in the circulation and have consequently been expected to be useful as drug-delivery systems. However, their applications have been limited because of the inability to introduce hydrophobic compounds to EVs without the use of harmful organic solvents. Herein, we developed an organic-solvent-free drug-loading technique based on the host exchange reaction. We demonstrated that the exchange reaction enabled quantitative loading of EVs with highly concentrated (0.1 mM) hydrophobic fullerene derivatives. Fullerene derivative-loaded EVs (EVs/C 60 ) could eliminate cancer cell lines more efficiently than fullerene derivative-loaded liposomes (Lip/C 60 ). Moreover, the photodynamic activity of EVs/C 60 was fivefold higher than that of the clinically available photosensitizer photofrin. EVs/C 60 could efficiently suppress tumor growth in tumor-xenograft model mice.

With their low immunogenicity and excellent deliverability, extracellular vesicles (EVs) are promising platforms for drug delivery systems. In this study, hydrophobic molecule loading techniques were developed via an exchange reaction based on supramolecular chemistry without using organic solvents that can induce EV disruption and harmful side effects. To demonstrate the availability of an exchanging reaction to prepare drug-loading EVs, hydrophobic boron cluster carborane (CB) was introduced to EVs (CB@EVs), which is expected as a boron agent for boron neutron capture therapy (BNCT). The exchange reaction enabled the encapsulation of CB to EVs without disrupting their structure and forming aggregates. Single-particle analysis revealed that an exchanging reaction can uniformly introduce cargo molecules to EVs, which is advantageous in formulating pharmaceuticals. The performance of CB@EVs as boron agents for BNCT was demonstrated in vitro and in vivo. Compared to L-BPA, a clinically available boron agent, and CB delivered with liposomes, CB@EV systems exhibited the highest BNCT activity in vitro due to their excellent deliverability of cargo molecules via an endocytosis-independent pathway. The system can deeply penetrate 3D cultured spheroids even in the presence of extracellular matrices. The EV-based system could efficiently accumulate in tumor tissues in tumor xenograft model mice with high selectivity, mainly via the enhanced permeation and retention effect, and the deliverability of cargo molecules to tumor tissues in vivo enhanced the therapeutic benefits of BNCT compared to the L-BPA/fructose complex. All of the features of EVs are also advantageous in establishing anticancer agent delivery platforms.

Background: Limited healing potential of the meniscus remains a burden for the successful repair of meniscus injuries in the orthopaedic fields. Silk-elastin (SE) is a novel recombinant protein with favorable properties for wound healing. This proof-of-concept study aimed to investigate the therapeutic effect of silk-elastin in a rabbit meniscal defect model., Methods: A migration assay using rabbit meniscus and synovial cells with various concentrations of SE in a culture medium was conducted to investigate the mechanism of meniscal healing by SE. Additionally, cylindrical defects with a 1.5 mm diameter were created at the anterior horn of the medial meniscus of rabbits. The animals were divided into three groups: 1) the Blank group; defect only, 2) the Col I group; implantation of type I atelocollagen sponge, and 3) the SE group; implantation of SE (150 mg/ml) sponge. Whole medial menisci were harvested at 4, 8, 12, and 24 weeks after surgery. Histological analyses including immunohistochemical staining were performed to assess meniscal healing., Results: In vitro study, Migration assay demonstrated a significantly higher number of migrated cells only in synovial cells. Especially, the SE concentration of 10 µg/mL demonstrated the highest number of migrated cells compared with other concentrations. In vivo study, the SE group exhibited significantly higher Ishida scores than other groups at all time points. Furthermore, the SE group showed higher synovial coverage scores than the Col I group at 4 and 8 weeks. Immunohistochemical staining demonstrated higher type II collagen staining in the SE group compared to other groups at 12 weeks. Implanted SE was efficiently replaced by safranin-O staining positive tissue within 8 weeks., Conclusions: SE could effectively repair a meniscal defect by inducing coverage of synovial cells. SE has the potential to be a useful material for meniscal repair., (© 2024. The Author(s).)

Objectives: Ageing and obesity are major risk factors for osteoarthritis (OA), a widespread disease currently lacking efficient treatments. Senescence-accelerated mouse prone 8 (SAMP8) display early onset ageing phenotypes, including OA. This study investigates the impacts of high-fat diet (HFD)-induced obesity on OA development in SAMP8., Methods: SAMP8 at 5 weeks were fed either a normal chow diet or an HFD for 10 weeks to induce obesity. Parameters related to obesity, liver function, and lipid and glucose metabolism were analysed. At 14 weeks of age, knee joint pathology, bone mineral density, and muscle strength were assessed. Immunohistochemistry and TUNEL staining were performed to evaluate markers for cartilage degeneration and chondrocyte apoptosis., Results: At 14 weeks of age, HFD-induced obesity increased liver and adipose tissue inflammation in SAMP8 without further exacerbating diabetes. Histological scoring revealed aggravated cartilage, menisci deterioration, and synovitis, while no further loss of bone mineral density or muscle strength was observed. Increased chondrocyte apoptosis was detected in knee joints following HFD feeding., Conclusions: Ten weeks of HFD feeding promotes spontaneous OA progression in 14-week-old SAMP8, potentially via liver damage that subsequently leads to chondrocyte apoptosis. This ageing-obese mouse model may prove valuable for further exploration of spontaneous OA pathophysiology., (© Japan College of Rheumatology 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Skeletal muscle injury (SMI) is often treated conservatively, although it can lead to scar tissue formation, which impedes muscle function and increases muscle re-injury risk. However, effective interventions for SMIs are yet to be established., Hypothesis: The administration of Silk Elastin® (SE), a novel artificial protein, to the SMI site can suppress scar formation and promote tissue repair., Study Design: A controlled laboratory study., Methods: In vitro : Fibroblast migration ability was assessed using a scratch assay. SE solution was added to the culture medium, and the fibroblast migration ability was compared across different concentrations. In vivo : An SMI model was established with Sprague-Dawley rats, which were assigned to three groups based on the material injected to the SMI site: SE gel (SE group; n = 8), atelocollagen gel (Atelo group; n = 8), and phosphate buffer saline (PBS group; n = 8). Histological evaluations were performed at weeks 1 and 4 following the SMI induction. In the 1-week model, we detected the expression of transforming growth factor (TGF)-β1 in the stroma using immunohistological evaluation and real-time polymerase chain reaction analysis. In the 4-week model, we measured tibialis anterior muscle strength upon peroneal nerve stimulation as a functional assessment., Results: In vitro : The fibroblast migration ability was suppressed by SE added at a concentration of 10⁴ μg/mL in the culture medium. In vivo : In the 1-week model, the SE group exhibited significantly lower TGFβ -1 expression than the PBS group. In the 4-week model, the SE group had a significantly larger regenerated muscle fiber diameter and smaller scar formation area ratio than the other two groups. Moreover, the SE group was superior to the other two groups in terms of regenerative muscle strength., Conclusion: Injection of SE gel to the SMI site may inhibit tissue scarring by reducing excessive fibroblast migration, thereby enhancing tissue repair., Clinical Relevance: The findings of this study may contribute to the development of an early intervention method for SMIs., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kyohei Nakata reports equipment, drugs, or supplies was provided by Hiroshima University Hospital. Silk elastin is provided by Sanyo Chemical Industries, Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Osteoarthritis (OA) is a common joint disorder characterized by cartilage degeneration, often leading to pain and functional impairment. Minced cartilage implantation (MCI) has emerged as a promising one-step alternative for large cartilage defects. However, the source of chondrocytes for MCI remains a challenge, particularly in advanced OA, as normal cartilage is scarce. We performed in vitro studies to evaluate the feasibility of MCI using osteophyte cartilage, which is present in patients with advanced OA. Osteophyte and articular cartilage samples were obtained from 22 patients who underwent total knee arthroplasty. Chondrocyte migration and proliferation were assessed using cartilage fragment/atelocollagen composites to compare the characteristics and regenerative potential of osteophytes and articular cartilage. Histological analysis revealed differences in cartilage composition between osteophytes and articular cartilage, with higher expression of type X collagen and increased chondrocyte proliferation in the osteophyte cartilage. Gene expression analysis identified distinct gene expression profiles between osteophytes and articular cartilage; the expression levels of COL2A1, ACAN, and SOX9 were not significantly different. Chondrocytes derived from osteophyte cartilage exhibit enhanced proliferation, and glycosaminoglycan production is increased in both osteophytes and articular cartilage. Osteophyte cartilage may serve as a viable alternative source of MCI for treating large cartilage defects in OA.

Microribonucleic acids (miRNAs) comprising miR-23a/b clusters, specifically miR-23a and miR-27a, are recognized for their divergent roles in myelination within the central nervous system. However, cluster-specific miRNA functions remain controversial as miRNAs within the same cluster have been suggested to function complementarily. This study aims to clarify the role of miR-23a/b clusters in myelination using mice with a miR-23a/b cluster deletion (KO mice), specifically in myelin expressing proteolipid protein (PLP). Inducible conditional KO mice were generated by crossing miR-23a/b cluster flox/flox mice with PlpCre-ERT2 mice; the offspring were injected with tamoxifen at 10 days or 10 weeks of age to induce a myelin-specific miR-23a/b cluster deletion. Evaluation was performed at 10 weeks or 12 months of age and compared with control mice that were not treated with tamoxifen. KO mice exhibit impaired motor function and hypoplastic myelin sheaths in the brain and spinal cord at 10 weeks and 12 months of age. Simultaneously, significant decreases in myelin basic protein (MBP) and PLP expression occur in KO mice. The percentages of oligodendrocyte precursors and mature oligodendrocytes are consistent between the KO and control mice. However, the proportion of oligodendrocytes expressing MBP is significantly lower in KO mice. Moreover, changes in protein expression occur in KO mice, with increased leucine zipper-like transcriptional regulator 1 expression, decreased R-RAS expression, and decreased phosphorylation of extracellular signal-regulated kinases. These findings highlight the significant influence of miR-23a/b clusters on myelination during postnatal growth and aging.

Aim: Osteoarthritis (OA) is a disease in which degeneration occurs in various tissues such as cartilage and subchondral bone. Degeneration of ligaments also plays an important role in OA progression, resulting in an increase in chondrocytes and ossification, but the factor that causes this is still unclear. It is reported that the expression of calcitonin gene-related peptide (CGRP) increases OA progression, and CGRP might play a role in ligament degeneration because CGRP has a function in endochondral ossification. The purpose of this study is to analyze the mechanism of ligament degeneration and the function of CGRP., Methods: To examine the relationship between ligament degeneration and CGRP expression, human posterior cruciate ligaments (PCL) from OA patients, and senescence-accelerated mouse prone 8 (SAMP8) mice were histologically analyzed. The effect of CGRP on human ligament cells on chondrogenesis, osteogenesis, and adipogenesis was also examined., Results: In human PCL and SAMP8 mice, CGRP expression increased as degeneration progressed, and decreased in severe degeneration. CGRP was expressed in the chondrocyte-like cells with SOX9. CGRP-positive cells expressing type II collagen increased with OA progression. CGRP upregulated the gene expression of VEGF, SOX9, RUNX2, COL10a1, and MMP13 in the human ligament cells. CGRP also promoted chondrogenesis and osteogenesis from the human ligament cells., Conclusion: During OA progression, CGRP plays a role in the transdifferentiation from ligament cells to chondrocytes and promotes endochondral ossification in the ligament. CGRP would be the therapeutic target to prevent ligament degeneration., (© 2023 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Suppressing inflammation and abnormal subchondral bone turnover is essential for reducing osteoarthritis (OA) progression and pain relief. This study focused on calcitonin gene-related peptide (CGRP), which is involved in inflammation and bone metabolism, and investigated whether a CGRP receptor antagonist (rimegepant) could suppress OA progression and relieve pain in two OA models. C57BL/6 mice (10-week-old) underwent surgical destabilization of the medial meniscus, and Rimegepant (1.0 mg/kg/100 μL) or phosphate-buffered saline (100 μL) was administered weekly intraperitoneally after OA surgery and evaluated at 4, 8, and 12 weeks. In the senescence-accelerated mice (SAM)-prone 8 (SAMP8), rimegepant was administered weekly before and after subchondral bone sclerosis and sacrificed at 9 and 23 weeks, respectively. Behavioral assessment and immunohistochemical staining (CGRP) of the dorsal root ganglion (DRG) were conducted to assess pain. In DMM mice, synovitis, cartilage degeneration, and osteosclerosis were significantly suppressed in the rimegepant group. In SAMP8, synovitis, cartilage degeneration, and osteosclerosis were significantly suppressed by rimegepant at 9 weeks; however, not at 23 weeks. Behavioral assessment shows the traveled distance and the number of standings in the rimegepant group were significantly longer and higher. In addition, CGRP expression of the DRG was significantly lower in the rimegepant group at 8 and 12 weeks of DMM and 9 weeks of SAMP8 treatment. No adverse effects were observed in either of the mouse models. Inhibition of CGRP signaling has the potential to be a therapeutic target to prevent OA progression and suppress pain through the attenuation of subchondral bone sclerosis and synovitis., (© 2023. Springer Nature Limited.)

Sepsis is a life-threatening inflammatory response to infection, often accompanied by skeletal muscle atrophy. A previous study demonstrated that the administration of microRNA-140 (miR-140) attenuated lipopolysaccharide (LPS)-induced muscle atrophy, whereas miR-140 knockdown with siRNA promoted atrophy. Therefore, we investigated whether miR-140 is involved in LPS-induced muscle atrophy using a genetic model, miR-140 -/- mice. We found that a single injection of LPS induced atrophy both in slow-twitch and fast-twitch muscles. The muscle weights and fiber cross-sectional areas were significantly reduced in both the wild-type (WT) and miR-140 -/- mice, with no difference between genotypes. The expression of several proteolysis markers, muscle-specific RING-finger 1 (MuRF1) and MAFbx/atrogin-1, increased in both groups after LPS injection. The ubiquitinated proteins in the miR-140 -/- mice were similar to those in the WT mice. Therefore, the deletion of miR-140 did not affect LPS-induced muscle atrophy. NEW & NOTEWORTHY We used miR-140 -/- mice to determine the function of miR-140 in LPS-induced skeletal muscle atrophy. To our knowledge, this study is the first to examine slow-twitch muscles in LPS-induced muscle wasting after miR-140 manipulation.

Background: Microfracture (MF) is frequently performed as a first-line treatment for articular cartilage defects. Although good clinical outcomes are often obtained in the short term, poor clinical outcomes sometimes occur because of subchondral bone deterioration. The condition of the subchondral bone treated with MF may affect the repair of the osteochondral unit., Purpose: To analyze histological findings of the osteochondral unit after performing MF on subchondral bone in different states-normal, absorption, and sclerosis-in a rat model., Study Design: Controlled laboratory study., Methods: Full-thickness cartilage defects (5.0 × 3.0 mm) were created in the weightbearing area of the medial femoral condyle in both knees of 47 Sprague-Dawley rats. Five MF holes were created within the cartilage defect using a 0.55-mm needle to a depth of 1 mm at 0 weeks (normal group), 2 weeks (absorption group), and 4 weeks (sclerosis group) after the cartilage defect was created. In the left knee, MF holes were filled with β-tricalcium phosphate (β-TCP). At 2 and 4 weeks after MF, knee joints were harvested and histologically analyzed., Results: MF holes were enlarged at 2 weeks and further enlarged at 4 weeks in all groups. In the absorption group, osteoclast accumulation around the MF holes and cyst formation were observed. The trabecular bone surrounding the MF holes was thickened in the sclerosis group. The diameter of the MF hole was largest in the absorption group at 2 and 4 weeks after MF compared with the other groups. No subchondral bone cysts were observed after β-TCP implantation. Pineda scores in all groups were significantly better with β-TCP implantation than without β-TCP implantation at 2 and 4 weeks., Conclusion: MF for subchondral bone with bone absorption induced enlargement of the MF holes, cyst formation, and delay of cartilage defect coverage. Implantation of β-TCP into the MF holes enhanced remodeling of the MF holes and improved repair of the osteochondral unit compared with MF only. Therefore, the condition of the subchondral bone treated with MF affects repair of the osteochondral unit in a cartilage defect.

Objective: Articular cartilage defect causes joint pain and finally progresses to osteoarthritis. Although the subchondral bone condition affects clinical outcomes of cartilage defects, the natural course of changes in subchondral bone and associated pain in full-thickness cartilage defects remain unknown. Therefore, we investigated the natural course of histological changes in subchondral bone and joint pain in cartilage defects using a rat model., Design: Full-thickness cartilage defects were created at the medial femoral condyle of 10-week-old male Sprague-Dawley rats. Rats were sacrificed at 3, 7, 14, 28, and 56 days postoperatively, and histological including immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining and micro-computed tomography (μCT) analyses of their knees were performed. Pain was evaluated using behavioral analysis and immunofluorescence staining of the dorsal root ganglion (DRG)., Results: The contour of the subchondral bone plate was maintained until day 3, but it was absorbed just under the cartilage defect from day 7 to 14. Starting on day 28, sclerotic changes surrounding the bone absorption area were detected. In the subchondral bone, the number of TRAP-positive cells peaked on day 14. Osteocalcin-positive cells were observed at 7 days, and their number gradually increased till day 56. Behavioral analysis showed that the total distance and the number of getting up by hind legs decreased on day 14. The number of calcitonin gene-related peptide-positive fibers in the DRG increased and was the highest on day 14., Conclusions: The subchondral bone condition under cartilage defects dynamically changes from bone resorption to sclerosis and is related to pain level.

Osteoarthritis (OA) is the most common age-related joint disease. However, the role of many microRNAs (miRNA) in skeletal development and OA pathogenesis has not been sufficiently elucidated using genetically modified mice with gain- and loss-of-function models. We generated Cartilage-specific miR-26a overexpressing (Col2a1-Cre;miR-26a Tg fl/fl : Cart-miR-26a Tg) mice and global miR-26a knockout (miR-26a KO) mice. The purpose of the present study was to determine the role of miR-26a in OA pathogenesis using aging and surgically induced models. Skeletal development of Cart-miR-26a Tg and miR-26a KO mice was grossly normal. Knee joints were evaluated by histological grading systems. In surgically-induced OA and aging models (12 and 18 months of age), Cart-miR-26a Tg mice and miR-26a KO mice exhibited OA-like changes such as proteoglycan loss and cartilage fibrillation with no significant differences in OARSI score (damage of articular cartilage) compared with control mice. However, miR-26a KO mice reduced muscle strength and bone mineral density at 12 months of age. These findings indicated that miR-26a modulates bone loss and muscle strength but has no essential role in aging-related or post-traumatic OA., Competing Interests: Conflict of Interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The prevalence of allergic disorders has increased worldwide in recent decades. Polyphenols, including resveratrol and curcumin, are posited to have potential as therapeutic agents for allergy; however, their use has been limited by poor water solubility. Accordingly, a highly concentrated, water dispersible, supramolecular complexes of polyphenols with polypeptides (poly-L-lysine, poly-γ-glutamic acid) and gelatin using high-speed vibration milling are developed. The complex exhibits resistance to photobleaching and thermal radiation. Treatment of a rat basophilic leukemia cell line (RBL-2H3) with polypeptide complexes containing resveratrol is suppressed allergic responses in vitro. Moreover, aerosolized administration of polypeptide complexes demonstrates excellent bioavailability and inhibition of immediate hypersensitivity reactions in ear tissue in vivo. Furthermore, the method avoids the use of organic solvent and therefore reduces undesirable biological responses., (© 2023 Wiley-VCH GmbH.)

Several microRNAs (miRNAs), including miR-23 and miR-27a have been reportedly involved in regulating myelination in the central nervous system. Although miR-23 and miR-27a form clusters in vivo and the clustered miRNAs are known to perform complementary functions, the role of these miRNA clusters in myelination has not been studied. To investigate the role of miR-23-27-24 clusters in myelination, we generated miR-23-27-24 cluster knockout mice and evaluated myelination in the brain and spinal cord. Our results showed that 10-week-old knockout mice had reduced motor function in the hanging wire test compared to the wild-type mice. At 4 weeks, 10 weeks, and 12 months of age, knockout mice showed reduced myelination compared to wild-type mice. The expression levels of myelin basic protein and myelin proteolipid protein were also significantly lower in the knockout mice compared to the wild-type mice. Although differentiation of oligodendrocyte progenitor cells to oligodendrocytes was not inhibited in the knockout mice, the percentage of oligodendrocytes expressing myelin basic protein was significantly lower in 4-week-old knockout mice than that in wild-type mice. Proteome analysis and western blotting showed increased expression of leucine-zipper-like transcription regulator 1 (LZTR1) and decreased expression of R-RAS and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the knockout mice. In summary, loss of miR-23-27-24 clusters reduces myelination and compromises motor functions in mice. Further, LZTR1, which regulates R-RAS upstream of the ERK1/2 pathway, a signal that promotes myelination, has been identified as a novel target of the miR-23-27-24 cluster in this study., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2023 Yuji Tsuchikawa et al.)

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation and is caused by various risk factors including aging and traumatic injury. Most microRNAs (miRNAs) have been associated with pathogenesis of osteoarthritis (OA) using in vitro models. However, the role of many miRNAs in skeletal development and OA pathogenesis is uncharacterized in vivo using genetically modified mice. Here, we focused on miR-23-27-24 clusters. There are two paralogous miR-23-27-24 clusters: miR-23a-27a-24-2 (miR-23a cluster) and miR-23b-27b-24-1 (miR-23b cluster). Each miR-23a/b, miR-24, and miR-27a/b is thought to function coordinately and complementary to each other, and the role of each miR-23a/b, miR-24, and miR-27a/b in OA pathogenesis is still controversial. MiR-23a/b clusters are highly expressed in chondrocytes and the present study examined their role in OA. We analyzed miRNA expression in chondrocytes and investigated cartilage-specific miR-23a/b clusters knockout (Col2a1-Cre; miR-23a/b flox/flox : Cart-miR-23clus KO) mice and global miR-23a/b clusters knockout (CAG-Cre; miR-23a/b flox/flox : Glob-miR-23clus KO) mice. Knees of Cart- and Glob-miR-23a/b clusters KO mice were evaluated by histological grading systems for knee joint tissues using aging model (12 and/or 18 month-old) and surgically-induced OA model. miR-23a/b clusters were among the most highly expressed miRNAs in chondrocytes. Skeletal development of Cart- and Glob-miR-23clus KO mice was grossly normal although Glob-miR-23clus KO had reduced body weight, adipose tissue and bone density. In the aging model and surgically-induced OA model, Cart- and Glob-miR-23clus KO mice exhibited mild OA-like changes such as proteoglycan loss and cartilage fibrillation. However, the histological scores were not significantly different in terms of the severity of OA in Cart- and Glob-miR-23clus KO mice compared with control mice. Together, miR-23a/b clusters, composed of miR-23a/b, miR-24, miR-27a/b do not significantly contribute to OA pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fujiwara, Ding, Sanada, Yimiti, Ishikawa, Nakasa, Kamei, Imaizumi, Lotz, Akimoto, Miyaki and Adachi.)

Purpose: Osteoarthritis (OA) is characterized by the degeneration of various tissues, including ligaments. However, pathological changes such as chondrogenesis and ossification in ligaments during OA are still unclear. Substance P (SP), a neuropeptide, has various functions including bone metabolism. This study aimed to analyze the expression and function of SP in OA ligaments, and the therapeutic potential of SP agonists in OA mice., Materials and Methods: Expressions of SP, SOX9, and MMP13 were histologically analyzed in the posterior cruciate ligament (PCL) in humans with OA and Senescence-accelerated mouse-prone 8 (SAMP8) mice as a spontaneous OA model. The effect of SP agonists on chondrogenesis was evaluated using human ligament cells. Finally, SP agonists were administered intraperitoneally to destabilized medial meniscus (DMM) mice, and the PCL was histologically evaluated., Results: In PCL of humans and mice, the expression of SP, SOX9, and MMP13 was upregulated as OA progressed, but their expression was downregulated in severe degeneration. SP and SOX9 were co-expressed in chondrocyte-like cells. In ligament cells, SP agonists downregulated SOX9, RUNX2, and COL10A1. On evaluating chondrogenesis in ligament cells, pellet diameter was reduced in those treated with the SP agonists compared to those untreated. Administration of SP agonists ameliorated PCL degeneration in DMM mice. The Osteoarthritis Research Society and ligament scores in mice with SP agonists were significantly lower than those without SP agonists., Conclusions: SP plays an important role in maintaining ligament homeostasis by inhibiting endochondral ossification during OA progression. Targeting SP has therapeutic potential for preventing ligament degeneration.

Background: Animal models of spontaneous osteoarthritis (OA) are sparse and not well characterized. The purpose of the present study is to examine OA-related changes and mechanisms in senescence-accelerated mouse prone 8 (SAMP8) that displays a phenotype of accelerated aging.  METHODS: Knees of male SAMP8 and SAM-resistant 1 (SAMR1) mice as control from 6 to 33 weeks of age were evaluated by histological grading systems for joint tissues (cartilage, meniscus, synovium, and subchondral bone), and µCT analysis. Gene expression patterns in articular cartilage were analyzed by real-time PCR. Immunohistochemistry was performed for OA-related factors, senescence markers, and apoptosis., Results: Starting at 14 weeks of age, SAMP8 exhibited mild OA-like changes such as proteoglycan loss and cartilage fibrillation. From 18 to 33 weeks of age, SAMP8 progressed to partial or full-thickness defects with exposure of subchondral bone on the medial tibia and exhibited synovitis. Histological scoring indicated significantly more severe OA in SAMP8 compared with SAMR1 from 14 weeks [median (interquartile range): SAMR1: 0.89 (0.56-1.81) vs SAMP8: 1.78 (1.35-4.62)] to 33 weeks of age [SAMR1: 1.67 (1.61-1.04) vs SAMP8: 13.03 (12.26-13.57)]. Subchondral bone sclerosis in the medial tibia, bone mineral density (BMD) loss of femoral metaphysis, and meniscus degeneration occurred much earlier than the onset of cartilage degeneration in SAMP8 at 14 weeks of age., Conclusions: SAMP8 are a spontaneous OA model that is useful for investigating the pathogenesis of primary OA and evaluating therapeutic interventions., (© 2022. The Author(s).)

Objectives: Substance P (SP) modulates NK1 and has various functions such as regulation of pain response, bone metabolism, and angiogenesis, which are recognized as important factors in osteoarthritis (OA). We aimed to evaluate the therapeutic effect of targeting SP on OA progression., Methods: SP expression patterns were analysed histologically in articular cartilage and subchondral bone of human knees from OA patients and autopsy donors as non-OA samples and in mouse articular cartilage. Moreover, to examine the effect of SP on the progression of OA, we administered drugs to mice following the surgical destabilization of the medial meniscus: Phosphate-buffered saline (PBS), septide (NK1 receptor agonist), or aprepitant (NK1 receptor antagonist). Histological analysis and bone morphologic analysis using micro-computed tomography were performed., Results: In human analysis, the expression of SP in mild OA samples was significantly higher than that in severe OA, and that in healthy cartilage was significantly higher than that in OA. In mouse analysis, Osteoarthritis Research Society International scores in the septide group were significantly lower than those in the control group. Computed tomography analysis showed that the subchondral bone's epiphysis in the control group had sclerotic change, not observed in the septide group., Conclusions: The administration of septide ameliorates OA progression through preventing subchondral bone sclerosis., (© Japan College of Rheumatology 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tendon is a fibrous connective tissue, that is, transmitting the forces that permit body movement. However, tendon/ligament biology is still not fully understood and especially, the role of miRNAs in tendon/ligament is sparse and uncharacterized in in vivo models. The objectives of this study were to address the function of DICER using mice with tendon/ligament-specific deletion of Dicer ( Dicer conditional knockout; cKO), and to identify key miRNAs in tendon/ligament. Dicer cKO mice exhibited hypoplastic tendons through structurally abnormal collagen fibrils with downregulation of tendon-related genes. The fragility of tendon did not significantly affect the tensile strength of tendon in Dicer cKO mice, but they showed larger dorsiflexion angle in gait compared with Control mice. We identified two miRNAs, miR-135a and miR-1247, which were highly expressed in the Achilles tendon of Control mice and were downregulated in the Achilles tendon of Dicer cKO mice compared with Control mice. miR-135a mimic increased the expression of tendon-related genes in injured Achilles tendon-derived fibroblasts. In this study, Dicer cKO mice exhibited immature tendons in which collagen fibrils have small diameter with the downregulation of tendon-related genes such as transcriptional factor, extracellular matrix, and miRNAs. Thus, DICER plays an important role in tendon maturation, and miR-135a may have the potential to become key miRNA for tendon maturation and healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Omoto, Yimiti, Sanada, Toriyama, Ding, Hayashi, Ikuta, Nakasa, Ishikawa, Sano, Lee, Akimoto, Shukunami, Miyaki and Adachi.)

The therapeutic potential of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) for various diseases and tissue repair is attracting attention. Here, EVs from conditioned medium of human bone marrow MSCs at passage 5 (P5) and passage 12 (P12) were analysed using mouse Achilles tendon rupture model and lectin microarray. P5 MSC-EVs accelerated Achilles tendon healing compared with P12 MSC-EVs. Fucose-specific lectin TJA-II was indicated as a glycan marker for therapeutic MSC-EVs. The present study demonstrated that early passaged MSC-EVs promote Achilles tendon healing compared with senescent MSC-EVs. Glycans on MSC-EVs might provide useful tools to establish a quality control and isolation system for therapeutic MSC-EVs in regenerative medicine., (© 2022 Federation of European Biochemical Societies.)

The chylous turbidity of blood samples is one of the causes of false-high hemoglobin (Hgb) concentration measurements by the colorimetric method, which has been widely applied in hematology analyzers. In such cases, additional manual procedures are required to correct Hgb concentrations. We therefore examined the effectiveness of an optical method for measuring Hgb concentrations in samples with chylous turbidity using Hgb-O in the reticulocyte channel equipped in XN-series analyzers (Sysmex, Kobe, Japan). Hgb-O showed excellent basic performance, including linear correlation and invariability with sodium lauryl sulfate (SLS)-Hgb detected by the colorimetric method. In the analysis of samples from healthy volunteers supplemented with fat emulsion, chylous turbidity did not affect Hgb-O but SLS-Hgb, which was falsely increased according to the dose of fat emulsion. Actually, SLS-Hgb was falsely elevated in 34 of 40 chylous turbidity 3+ samples. The remaining 6 samples were measured in hematology analyzers where Hgb-O was inconsistent with SLS-Hgb in the internal quality control records. For these samples, the correction factors calculated from the internal quality control records could contribute to providing the corrected Hgb-O value. These findings suggested that the optical method was effective and convenient for accurately evaluating Hgb concentrations in samples with extremely chylous turbidity., (© 2021. The Author(s).)

Osteoarthritis (OA) is a common aging-associated disease that clinically manifests as joint pain, mobility limitations, and compromised quality of life. Today, OA treatment is limited to pain management and joint arthroplasty at the later stages of disease progression. OA pathogenesis is predominantly mediated by oxidative damage to joint cartilage extracellular matrix and local cells such as chondrocytes, osteoclasts, osteoblasts, and synovial fibroblasts. Under normal conditions, cells prevent the accumulation of reactive oxygen species (ROS) under oxidatively stressful conditions through their adaptive cytoprotective mechanisms. Heme oxygenase-1 (HO-1) is an iron-dependent cytoprotective enzyme that functions as the inducible form of HO. HO-1 and its metabolites carbon monoxide and biliverdin contribute towards the maintenance of redox homeostasis. HO-1 expression is primarily regulated at the transcriptional level through transcriptional factor nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), specificity protein 1 (Sp1), transcriptional repressor BTB-and-CNC homology 1 (Bach1), and epigenetic regulation. Several studies report that HO-1 expression can be regulated using various antioxidative factors and chemical compounds, suggesting therapeutic implications in OA pathogenesis as well as in the wider context of joint disease. Here, we review the protective role of HO-1 in OA with a focus on the regulatory mechanisms that mediate HO-1 activity.

Objective: Autologous chondrocyte implantation is a necessary procedure for the repair of articular cartilage defects; however, isolated chondrocyte implantation requires a 2-step procedure (for harvesting and implantation) and is limited by cytotoxicity due to enzymatic digestion. Therefore, in this in vitro study, we evaluated the possible benefit of using minced cartilage embedded in a 3-dimensional culture scaffold and fixed with fibrin glue, in comparison with isolated chondrocytes in atelocollagen, to induce cell migration, proliferation, and matrix production, using cartilage from patients with knee joint osteoarthritis., Design: Cartilage fragments were obtained from 7 female patients with knee osteoarthritis (OA) and embedded in atelocollagen gels. As a control, chondrocytes were isolated and embedded in gels in the same manner. These composites were cultured for 3 weeks, and cell proliferation and matrix production were evaluated using histology and immunochemistry., Results: Histologically, minced cartilage showed cell migration from the cartilage fragments into the gel, with the Bern score and cell count in the minced cartilage group being significantly higher than those in the control group. Immunohistochemistry revealed that the number of Ki67-positive cells, the expression of LECT-1 and TGF-β, and the glycosaminoglycan content were significantly higher in the minced cartilage than in the control group. Minced cartilage exhibited superior cell migration, proliferation, and glycosaminoglycan content than isolated chondrocytes., Conclusion: Our findings support that minced cartilage has a favorable potential for cell proliferation and matrix production compared with the isolated chondrocytes after enzymatic treatment.

Context: Hot-spring therapy is occasionally used for the treatment of inflammatory diseases. Microorganisms might contribute to the anti-inflammatory functions seen in thermal mud therapies. Natural microorganisms, derived from traditional spa resorts, could be useful as a preventive strategy for alternative medical applications., Objective: The aim of the study was to find effective microalgae from prominent hot springs to use for the treatment of inflammatory diseases., Design: The research team performed an in-vitro study. Microalgae, derived from Beppu hot springs, were isolated and homogeneously cultured., Setting: The study took place at the Saravio Central Institute at Saravio Cosmetics in Oita, Japan and the Department of Bioscience and Biotechnology in the Graduate School of Agriculture at Shinshu University in Nagano, Japan., Intervention: For identification, the 18S ribosomal RNA genes of microalgae were investigated by DNA sequencing and homology search, together with microscopic observation., Outcome Measures: To examine the pharmacological activities of the algal extracts, real-time polymerase chain reactions were performed, using either primary dermal fibroblasts (DFs), dermal papilla cells (DPCs), or fibroblast-like synoviocytes (FLSs). To test the antioxidant activity, both the oxygen radical absorbance capacity and the generation of intracellular reactive oxygen species (ROS) were evaluated., Results: A novel strain of green algae, Mucidosphaerium sp., was isolated from a Beppu hot spring. The algal extract downregulated gene-expression levels of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor- alpha (TNF-α), in various primary cells pre-exposed to IL-1β. The protein level of the risk factors was concomitantly reduced. In addition, the algal extract suppressed the IL-1β-induced upregulation of cyclooxygenase-2, nerve growth factor, and matrix metalloproteinase-1 (MMP-1) and MMP-3 in DFs. It also inhibited that of MMP-1, -3, and -9 in FLSs. Moreover, the extract inhibited total MMP protease activities. The microalgae decreased the intracellular reactive oxygen species (ROS) level in FLSs with an antioxidant activity of 178.3 ± 0.9 μmol of trolox equivalent/g., Conclusions: The present study showed that the novel Mucidosphaerium sp., derived from a Beppu hot spring, suppressed inflammatory reactions in both cutaneous and articular cells, partly due to its antioxidative properties. The novel algal strain may be a useful tool as an alternative medicine for skin and joint inflammatory disorders.

Upstream open reading frames (uORFs) are present in the 5'-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1517 (1373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.