In vivo therapy of osteosarcoma using anion transporters-based supramolecular drugs.

BACKGROUND: Osteosarcoma represents a serious clinical challenge due to its widespread genomic alterations, tendency for drug resistance and distant metastasis. New treatment methods are urgently needed to address those treatment difficulties in osteosarcoma to improve patient prognoses. In recent years, small-molecule based anion transporter have emerged as innovative and promising therapeutic compound with various biomedical applications. However, due to a lack of efficient delivery methods, using ion transporters as therapeutic drugs in vivo remains a major challenge. RESULT: Herein, we developed self-assembled supramolecular drugs based on small-molecule anion transporters, which exhibited potent therapeutic effect towards osteosarcoma both in vitro and in vivo. The anion transporters can disrupt intracellular ion homeostasis, inhibit proliferation, migration, epithelial-mesenchymal transition process, and lead to osteosarcoma cell death. RNA sequencing, western blot and flow cytometry indicated reprogramming of HOS cells and induced cell death through multiple pathways. These pathways included activation of endoplasmic reticulum stress, autophagy, apoptosis and cell cycle arrest, which avoided the development of drug resistance in osteosarcoma cells. Functionalized with osteosarcoma targeting peptide, the assembled supramolecular drug showed excellent targeted anticancer therapy against subcutaneous xenograft tumor and lung metastasis models. Besides good tumor targeting capability and anti-drug resistance, the efficacy of the assembly was also attributed to its ability to regulate the tumor immune microenvironment in vivo. CONCLUSIONS: In summary, we have demonstrated for the first time that small-molecule anion transporters are capable of killing osteosarcoma cells through multiple pathways. The assemblies, OTP-BP-L, show excellent targeting and therapeutic effect towards osteosarcoma tumors. Furthermore, the supramolecular drug shows a strong ability to regulate the tumor immune microenvironment in vivo. This work not only demonstrated the biomedical value of small-molecule anion transporters in vivo, but also provided an innovative approach for the treatment of osteosarcoma.

Injectable hydrogel encapsulating siMMP13 with anti-ROS and anti-apoptotic functions for osteoarthritis treatment.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive degeneration of articular cartilage, leading to pain, stiffness, and loss of joint function. The pathogenesis of OA involves multiple factors, including increased intracellular reactive oxygen species (ROS), enhanced chondrocyte apoptosis, and disturbances in cartilage matrix metabolism. These processes contribute to the breakdown of the extracellular matrix (ECM) and the loss of cartilage integrity, ultimately resulting in joint damage and dysfunction. RNA interference (RNAi) therapy has emerged as a promising approach for the treatment of various diseases, including hATTR and acute hepatic porphyria. By harnessing the natural cellular machinery for gene silencing, RNAi allows for the specific inhibition of target genes involved in disease pathogenesis. In the context of OA, targeting key molecules such as matrix metalloproteinase-13 (MMP13), which plays a critical role in cartilage degradation, holds great therapeutic potential. RESULTS: In this study, we developed an innovative therapeutic approach for OA using a combination of liposome-encapsulated siMMP13 and NG-Monomethyl-L-arginine Acetate (L-NMMA) to form an injectable hydrogel. The hydrogel served as a delivery vehicle for the siMMP13, allowing for sustained release and targeted delivery to the affected joint. Experiments conducted on destabilization of the medial meniscus (DMM) model mice demonstrated the therapeutic efficacy of this composite hydrogel. Treatment with the hydrogel significantly inhibited the degradation of cartilage matrix, as evidenced by histological analysis showing preserved cartilage structure and reduced loss of proteoglycans. Moreover, the hydrogel effectively suppressed intracellular ROS accumulation in chondrocytes, indicating its anti-oxidative properties. Furthermore, it attenuated chondrocyte apoptosis, as demonstrated by decreased levels of apoptotic markers. CONCLUSION: In summary, the injectable hydrogel containing siMMP13, endowed with anti-ROS and anti-apoptotic properties, may represent an effective therapeutic strategy for osteoarthritis in the future.

Spatial Transcriptomics of Aging Rat Ovaries Reveals Unexplored Cell Subpopulations with Reduced Antioxidative Defense.

INTRODUCTION: Ovarian aging is characterized by a gradual decline in quantity and quality of oocytes and lower chance of fertility. Better understanding the genetic modulation during ovarian aging can further address available treatment options for aging-related ovarian diseases and fertility preservation. METHODS: A novel technique spatial transcriptomics (ST) was used to investigate the spatial transcriptome features of rat ovaries. Transcriptomes from ST spots in the young and aged ovaries were clustered using differentially expressed genes. These data were analyzed to determine the spatial organization of age-induced heterogeneity and potential mechanisms underlying ovarian aging. RESULTS: In this study, ST technology was applied to profile the comprehensive spatial imaging in young and aged rat ovary. Fifteen ovarian cell clusters with distinct gene-expression signatures were identified. The gene expression dynamics of granulosa cell clusters revealed three sub-types with sequential developmental stages. Aged ovary showed a significant decrease in the number of granulosa cells from the antral follicle. Besides, a remarkable rearrangement of interstitial gland cells was detected in aging ovary. Further analysis of aging-associated transcriptional changes revealed that the disturbance of oxidative pathway was a crucial factor in ovarian aging. CONCLUSIONS: This study firstly described an aging-related spatial transcriptome changes in ovary and identified the potential targets for prevention of ovarian aging. These data may provide the basis for further investigations of the diagnosis and treatment of aging-related ovarian disorders.

Osteal Tissue Macrophages Are Involved in Endplate Osteosclerosis through the OSM-STAT3/YAP1 Signaling Axis in Modic Changes.

Modic changes (MCs) are radiographic manifestations of lumbar degenerative diseases. Various types of MCs are often associated with endplate osteosclerosis. Osteal tissue macrophages (Osteomacs) were reported to be crucial for bone homeostasis and bone repair, but whether osteomacs participate in the endplate osteosclerosis in MCs remained unclear. In this study, we tried to explore the critical role of osteomacs in regulating osteogenesis in MCs. We collected MCs from patient samples and developed a Propionibacterium acnes-induced rat MCs model, using microcomputed tomography and immunohistochemistry to detect the endplate bone mass and distribution of osteomacs. In patients' MCs, osteomacs increased in endplate subchondral bone, especially in Modic type II. Endplate in Modic type III presented a stable osteosclerosis. In rat MCs model, osteomacs increased in the bone hyperplasia area but not in the inflammation area of the endplate region, whereas the distribution of osteomacs was consistent with the area of osteosclerosis. To further explore the functions of osteomacs in vitro, we isolated osteomacs using MACS technology and found osteomacs secreted oncostatin M (OSM) and strongly promoted osteoblast differentiation rather than osteoclast through the mechanism of OSM-mediated tyrosine phosphorylation and interaction of STAT3 and Yes-associated protein 1 (YAP1). STAT3 phosphorylation inhibition or YAP1 knockdown attenuated OSM-mediated osteoblast differentiation. Finally, we confirmed that blockade of OSM in vivo using anti-OSM-neutralizing Ab prevented endplate osteosclerosis in rat MCs model. Taken together, these findings confirmed that endplate osteosclerosis in MCs was accompanied by an increased number of osteomacs, which regulated osteogenesis via the OSM-STAT3/YAP1 signaling axis.

Learning-based fully automated prediction of lumbar disc degeneration progression with specified clinical parameters and preliminary validation.

BACKGROUND: Lumbar disc degeneration (LDD) may be related to aging, biomechanical and genetic factors. Despite the extensive work on understanding its etiology, there is currently no automated tool for accurate prediction of its progression. PURPOSE: We aim to establish a novel deep learning-based pipeline to predict the progression of LDD-related findings using lumbar MRIs. MATERIALS AND METHODS: We utilized our dataset with MRIs acquired from 1,343 individual participants (taken at the baseline and the 5-year follow-up timepoint), and progression assessments (the Schneiderman score, disc bulging, and Pfirrmann grading) that were labelled by spine specialists with over ten years clinical experience. Our new pipeline was realized by integrating the MRI-SegFlow and the Visual Geometry Group-Medium (VGG-M) for automated disc region detection and LDD progression prediction correspondingly. The LDD progression was quantified by comparing the Schneiderman score, disc bulging and Pfirrmann grading at the baseline and at follow-up. A fivefold cross-validation was conducted to assess the predictive performance of the new pipeline. RESULTS: Our pipeline achieved very good performances on the LDD progression prediction, with high progression prediction accuracy of the Schneiderman score (Accuracy: 90.2 ± 0.9%), disc bulging (Accuracy: 90.4% ± 1.1%), and Pfirrmann grading (Accuracy: 89.9% ± 2.1%). CONCLUSION: This is the first attempt of using deep learning to predict LDD progression on a large dataset with 5-year follow-up. Requiring no human interference, our pipeline can potentially achieve similar predictive performances in new settings with minimal efforts.

Poor Bone Quality, Multilevel Surgery, and Narrow and Tall Cages Are Associated with Intraoperative Endplate Injuries and Late-onset Cage Subsidence in Lateral Lumbar Interbody Fusion: A Systematic Review.

BACKGROUND: A major complication of lateral lumbar interbody fusion (LLIF) is cage subsidence, which may lead to clinical problems, including loss of disc height correction, altered spinal alignment, recurrent pain, and vertebral body fracture. A thorough review of the current knowledge about the risk factors for the two types of cage subsidence after LLIF-intraoperative endplate injury and late-onset cage subsidence-could bring attention to well-established risk factors for clinical consideration while identifying any incompletely characterized factors that require further research to clarify. QUESTIONS/PURPOSES: We performed a systematic review to answer the following questions: (1) Are bone quality and surrogates for bone quality, such as patient age and sex, associated with an increased likelihood of cage subsidence? (2) Are implant-related factors associated with an increased likelihood of cage subsidence? METHODS: Two independent reviewers comprehensively searched Medline, Embase, Cochrane Library, PubMed, and Web of Science from 1997 to 2020 to identify all potential risk factors for cage subsidence after LLIF. Discrepancies were settled through discussion during full-text screening. Search terms included "lateral" AND "interbody fusion" AND "subsidence" OR "settling" OR "endplate injury" OR "endplate violation" WITHOUT "cervical" OR "transforaminal" OR "biomechanical." Eligible studies were retrospective or prospective comparative studies, randomized controlled trials, and case series with sample sizes of 10 patients or more reporting risk factors for cage subsidence or endplate injury after LLIF. Studies that involved cervical interbody fusions and biomechanical and cadaveric experiments were excluded. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to assess the studies' quality of evidence. The initial database review found 400 articles. Thirty-four articles with moderate- to very-low-quality evidence met the inclusion criteria for analysis. A total of 3233 patients (58% [1860] of whom were female) were included in this review. Two types of cage subsidence were reviewed: late-onset cage subsidence, which occurs gradually postoperatively, and intraoperative endplate injury, which is derived from iatrogenic endplate violation during endplate preparation or cage insertion. Among 20 studies with moderate quality of evidence according to the GRADE criteria, eight studies reported risk factors for cage subsidence related to bone mineral density and its surrogates and 12 studies focused on risk factors regarding implant factors, including cage dimension, cage material, construct length, and supplementary instrumentation. RESULTS: Patients with a dual x-ray absorptiometry T-score of -1.0 or less, age older than 65 years, and female sex were considered to have a high risk of both types of cage subsidence. Regarding cage size, cage width ≥ 22 mm helped to avoid late-onset cage subsidence, and cage height ≤ 11 mm was recommended by some studies to avoid intraoperative endplate injuries. Studies recommended that multilevel LLIF should be conducted with extra caution because of a high risk of losing the effect of indirect decompression. Studies found that standalone LLIF might be sufficient for patients without osteoporosis or obesity, and supplementary instrumentation should be considered to maintain the postoperative disc height and prevent subsidence progression in patients with multiple risk factors. The effect of the bone graft, cage material, endplate condition, and supplementary instrumentation on cage subsidence remained vague or controversial. CONCLUSION: Patients with poor bone density, patients who are older than 65 years, and female patients should be counseled about their high risk of developing cage subsidence. Surgeons should avoid narrow cages when performing LLIF to minimize the risk of late-onset cage subsidence, while being cautious of an aggressive attempt to restore disc height with a tall cage as it may lead to intraoperative endplate injury. For multilevel constructs, direct decompression approaches, such as posterior and transforaminal LIF, should be considered before LLIF, since the effect of indirect decompression may be difficult to maintain in multilevel LLIF because of high risks of cage subsidence. The effect of the cage material and supplementary instrumentation require stronger evidence from prospectively designed studies with larger sample size that randomly assign patients to polyetheretherketone (PEEK) or titanium cages and different fixation types. Future research on intraoperative endplate injuries should focus on the specific timing of when endplate violation occurs with the help of intraoperative imaging so that attempts can be made to minimize its occurrence. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Methyl 3,4-dihydroxybenzoate inhibits RANKL-induced osteoclastogenesis via Nrf2 signaling in vitro and suppresses LPS-induced osteolysis and ovariectomy-induced osteoporosis in vivo.

Osteoporosis deteriorates bone mass and biomechanical strength and is life-threatening to the elderly. In this study, we show that methyl 3,4-dihydroxybenzoate (MDHB), an antioxidant small-molecule compound extracted from natural plants, inhibits receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro. Furthermore, MDHB attenuates the activation of mitogen-activated protein kinase (MAPK) and NF-κB pathways by reducing the levels of reactive oxygen species (ROS), which leads to downregulated protein expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1). We also confirm that MDHB upregulates the protein expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), an important transcription factor involved in ROS regulation, by inhibiting the ubiquitination-mediated proteasomal degradation of Nrf2. Next, animal experiments show that MDHB has an effective therapeutic effect on lipopolysaccharide (LPS)- and ovariectomized (OVX)-induced bone loss in mice. Our study demonstrates that MDHB can upregulate Nrf2 and suppress excessive osteoclast activity in mice to treat osteoporosis.

Damage to the human lumbar cartilage endplate and its clinical implications.

The cartilaginous endplate (CEP) is a thin layer of hyaline cartilage, and plays an important role in the diffusion of nutrients into the intervertebral discs. Its damage may seriously affect the disc degeneration, and result in low back pain (LBP). However, the structural features of damaged CEPs have not been well characterized, and this hinders our understanding of the etiology of disc degeneration and pain. To present the structural features of micro-damaged CEPs in patients with disc degeneration and LBP that might even be regarded as an initial factor for disc degeneration, we performed a histological study of micro-damaged CEPs harvested from human lumbar intervertebral discs and analyzed its clinical implications. Human lumbar CEPs were excised from 35 patients (mean age 60.91 years) who had disc degeneration and LBP. Control tissue was obtained from 15 patients (mean age 54.67 years) with lumbar vertebral burst fractures. LBP and disability were assessed clinically, and all patients underwent anterior vertebral body fusion surgery. CEPs together with some adjacent nucleus pulposus (NP) were sectioned at 4 µm, and stained using H&E, Safranin O/Fast Green, and Alcian Blue. Immunostaining and PCR were used to identify various markers of degeneration, innervation, and inflammation. Histology demonstrated physical micro-damage in 14/35 CEPs from the disc degeneration group. Six major types of damage could be distinguished: fissure, traumatic nodes, vascular mimicry, incorporation of NP tissue within the CEP, incorporation of bone within the CEP, and incorporation of NP and bone within the CEP. Pain and disability scores (ODI: p = 0.0190; JOA: p = 0.0205; JOABPEQ: p = 0.0034) were significantly higher in those with micro-damaged CEPs (N = 14) than in those with non-damaged CEPs (N = 21). CEP damage was significantly associated with elevated MMP3 (p = 0.043), MMP13 (p = 0.0191), ADAMTS5 (p = 0.0253), TNF-α (p = 0.0011), and Substance P (p = 0.0028), and with reduced Sox9 (p = 0.0212), aggrecan (p = 0.0127), and type II collagen (p = 0.0139). In conclusion, we presented a new classification of human lumbar micro-damaged CEPs. Furthermore, we verify disc degeneration, innervation, and discogenic pain in micro-damaged CEPs.

Importance of the epiphyseal ring in OLIF stand-alone surgery: a biomechanical study on cadaveric spines.

PURPOSES: To explore the function of endplate epiphyseal ring in OLIF stand-alone surgery using a biomechanical model to reduce the complications of endplate collapse and cage subsidence. METHODS: In total, 24 human cadaveric lumbar function units (L1-2 and L3-4 segments) were randomly assigned to two groups. The first group was implanted with long fusion cages which engaged with both inner and outer regions of epiphyseal ring (Complete Span-Epiphyseal Ring, CSER). Those engaged with only the inner half of epiphyseal ring were the second group (Half Span-Epiphyseal Ring, HSER). Each group was divided into two subgroups [higher cage-height (HH) and normal cage-height (NH)]. Specimens were fixed in testing cups and compressed at approximately 2.5 mm/s, until the first sign of structural failure. Trabecular structural damage was analyzed by Micro-CT, as well as the difference of bone volume fraction (BV/TV), trabecular thickness (Tb.Th) et al. in different regions. RESULTS: Endplate collapse was mainly evident in the inner region of epiphyseal ring, where trabecular injury of sub-endplate bone was most concentrated. Endplate collapse incidence was significantly higher in HSER than CSER specimens (P = 0.017). A structural failure occurred at a lower force in HSER (1.41 ± 0.34 KN) compared with CSER (2.44 ± 0.59 KN). HH subgroups failed at a lower average force than NH subgroups. Micro-CT results showed a more extensive trabecular fracture in HSER specimens compared to CSER specimens, especially in HH subgroup. CONCLUSIONS: Endplate collapse is more likely to occur with short half span cages than complete span cages, and taller cages compared with normal height cages. During OLIF surgery, we should choose cages matching intervertebral disc space height and place the cages spanning over the whole epiphyseal ring to improve support strength.

The correlation between the change of Hounsfield units value and Modic changes in the lumbar vertebral endplate.

OBJECTIVES: To evaluate the changes of Hounsfield units (HU) value in different types of Modic changes (MCs) and to analyze the correlation between the change of HU value and area ratio of MCs region, bone mineral density (BMD), and degree of intervertebral disc degeneration. METHODS: One hundred fifty-eight endplates with MCs were included and analyzed. HU values of MCs regions and adjacent vertebral corresponding regions without MCs were measured. The area ratio of MCs region was defined as the area of MCs divided by the area of endplate or the vertebral sagittal plane. BMD was measured by Dual-energy x-ray absorptiometry (DXA). Degree of intervertebral disc degeneration was evaluated based on Pfirrmann classification. According to the types of variables, descriptive statistics, Kolmogorove-Smirnov test, paired t-test, Wilcoxon signed-rank test, Independent-Samples T Test, and Pearson correlation analysis were used. RESULTS: The HU values in any types of MCs are significantly higher than that of adjacent vertebral corresponding regions without MCs (P < 0.001). The HU value of the type III MCs is higher than that of the type I and type II MCs. HU value was positively correlated with BMD. In the levels with Grade V disc degeneration, the area ratio of MCs region was significant increased. CONCLUSIONS: HU values of the vertebral endplate and bone marrow were increased in most MCs regions with all types of MCs. HU value of endplates had a significantly positive correlation with BMD. Higher area ratio of MCs region is associated with more severe intervertebral disc degeneration.

IL-6/YAP1/ß-catenin signaling is involved in intervertebral disc degeneration.

Yes-associated protein 1 (YAP1) is a transcriptional coactivator and negative regulator of the Hippo pathway. It regulates diverse cellular processes, such as cell proliferation, contact inhibition, and tissue size. However, the role of YAP1 in intervertebral disc degeneration (IDD) remains elusive. Here, we demonstrated that YAP1 was activated by Interleukin 6 (IL-6) through tyrosine phosphorylation in nucleus pulposus cells (NP cells). Overexpression of YAP1 decreased Sox-9, Col-II, aggrecan expression, whereas increased matrix metalloproteinases 13 level. In contrast, knockdown of YAP1 by small interfering RNA (siRNA) showed opposite effects and rescued IL-6 induced NP cells degeneration. In addition, western blot showed that IL-6 treatment increased YAP1 and ß-catenin protein level; co-immunoprecipitation (Co-IP) and immunofluorescence analysis showed that IL-6 enhanced YAP1 and ß-catenin interaction and nuclear accumulation. Knockdown of ß-catenin by siRNA blocked IL-6 treatment or YAP1 overexpression induced degeneration. Moreover, we found that verteporfin, a specific inhibitor of YAP1, effectively alleviated IDD development in rat disks. Taken together, our findings indicated that YAP1 plays an important role in IDD, and ß-catenin is essential for IL-6/YAP1 signaling.

Painful Schmorl's nodes treated by discography and discoblock.

PURPOSE: To determine the efficacy of discography and discoblock in the treatment of low back pain (LBP) associated with painful Schmorl's nodes (SNs). METHODS: Between January 2010 and February 2015, 46 patients were studied who had LBP suspected to be secondary to SNs. There were 34 men and 12 women, and mean age was 54.2 years (range 42-68 years). All patients underwent provocation discography, and discoblock was given to positive patients (confirmed to have painful SNs). Visual analogue scores (VAS) and the Oswestry Disability Index (ODI) were evaluated at 4 h and 1, 3, 6, and 12 months post-operatively. MRI was also used to evaluate the SNs at 12 months. RESULTS: Discography was performed on a total of 60 discs without infection or other complications. Positive findings were found in 71.7% discs; 20.0% were negative, and 8.3% were indeterminate. Among the positive patients who underwent discoblock, 89.2% reported an improvement in their LBP, and none reported worsening symptoms. VAS and ODI scores decreased significantly after discoblock, and there were no significant differences between 4 h and 1, 3, 6, and 12 months post-operatively. In patients with painful SNs, the vertebral body bone marrow surrounding the SN was characterized by low T1 and high T2 signals on MRI. At 12 months, the node demonstrated either high T1 and T2 signals or low T1 and T2 signals. The SNs tended to remain stable in size over time. CONCLUSIONS: Painful SNs refractory to medical or physical therapy should be an indication for treatment with discography and discoblock.

Low virulence bacterial infections in cervical intervertebral discs: a prospective case series.

STUDY DESIGN: A prospective cross-sectional case series study. OBJECTIVE: To investigate the prevalence of low virulence disc infection and its associations with characteristics of patients or discs in the cervical spine. BACKGROUND: Low virulence bacterial infections could be a possible cause of intervertebral disc degeneration and/or back pain. Controversies are continuing over whether these bacteria, predominantly Propionibacterium acnes (P. acnes), represent infection or contamination. However, the current studies mainly focus on the lumbar spine, with very limited data on the cervical spine. METHODS: Thirty-two patients (20 men and 12 women) who underwent anterior cervical fusion for degenerative cervical spondylosis or traumatic cervical cord injury were enrolled. Radiological assessments included X-ray, CT, and MRI of the cervical spine. Endplate Modic changes, intervertebral range of motion, and disc herniation type were evaluated. Disc and muscle tissues were collected under strict sterile conditions. Samples were enriched in tryptone soy broth and subcultured under anaerobic conditions, followed by identification of the resulting colonies by the PCR method. RESULTS: Sixty-six intervertebral discs were excised from thirty-two patients. Positive disc cultures were noted in eight patients (25%) and in nine discs (13.6%). The muscle biopsy (control) cultures were negative in 28 patients and positive in 4 patients (12.5%); three of whom had a negative disc culture. Seven discs (10.6%) were positive for coagulase-negative Staphylococci (CNS) and two discs were positive for P. acnes (3.0%). A younger patient age and the extrusion or sequestration type of disc herniation, which represented a complete annulus fibrous failure, were associated with positive disc culture. CONCLUSIONS: Our data show that CNS is more prevalent than P. acnes in degenerative cervical discs. The infection route in cervical discs may be predominantly through an annulus fissure. Correlation between these infections and clinical symptoms is uncertain; therefore, their clinical significance needs to be investigated in the future. These slides can be retrieved under Electronic Supplementary Material.

Does the high-intensity zone (HIZ) of lumbar Intervertebral discs always represent an annular fissure?

OBJECTIVES: The aim of this study was to examine high-intensity zone (HIZ) characteristics on both T1- and T2-weighted sagittal magnetic resonance (MR) images, and to reveal their exact nature. MATERIALS: Seventy-three patients with low back pain and HIZs (identified on T2-weighted images) were included. Patients, aged 25-80 years (mean 51), were divided into two groups: the 'single-HIZ' group exhibited HIZs only on T2-weighted images, while the 'dual-HIZ' group exhibited HIZs on both T2-weighted and T1-weighted images. Tissue corresponding to the HIZ was harvested from surgery for analysis. RESULTS: Eighty-two discs were studied, from 39 patients with single HIZs, 30 with dual HIZs, and four with both in the posterior annulus. HIZ volume, volume ratio, and signal intensity on T2-weighted images from the dual-HIZ group were significantly greater. Surgery was able to successfully restore patients' ability in both groups, while conservative treatments were less effective for patients with dual HIZs. Histology revealed outer annular fissures invaded by granulation tissue in the single-HIZ group. In dual-HIZ discs, Von Kossa staining and CT scans showed more calcified or ossified lesions (94.1 vs. 0 %, P<0.001), and chemical analysis showed significantly higher calcium content. CONCLUSIONS: HIZs on both T2- and T1-weighted images represent calcified tissue, possibly from a vertebral endplate. A new concept of dual HIZ should be defined. KEY POINTS: • Conventional definition of an HIZ refers to T2-weighted images only. • Dual HIZs have greater HIZ volume, volume ratio, and signal intensity. • HIZs on both T2- and T1-weighted images represent calcified tissue. • Conservative treatments are less likely to be effective for patients with dual HIZs.

Effect of pure muscle retraction on multifidus injury and atrophy after posterior lumbar spine surgery with 24 weeks observation in a rabbit model.

PURPOSE: To evaluate the effect of pure muscle retraction on multifidus injury and atrophy. MATERIALS AND METHODS: Sixty-three adult New Zealand white rabbits were divided evenly into three groups: 1-h retraction (group R1), 2-h retraction (R2), and sham surgery (C). The multifidus muscle was evaluated using magnetic resonance imaging (MRI) and histology after 3 and 48 h, and 1, 3, 6, 12, and 24 weeks after surgery. RESULTS: Multifidus muscle injury and atrophy were not observed in group C, but were obvious in groups R1 and R2. Edema, necrosis, and inflammation mainly occurred in the first week postoperatively, and were more severe in R2 than in R1 (P < 0.01). Muscle fiber regeneration began at week 1, fibrotic changes mainly occurred at weeks 3 and 6, and fat degeneration became obvious at weeks 12 and 24 postoperatively. The fibrosis and fat degeneration scores of R2 were higher than those of R1 (P < 0.01). Decreased acetylcholine activity and granular degeneration of the neuromuscular junction were observed in both retraction groups, but was more severe in R2 than in R1 (P < 0.01). CONCLUSION: Muscle retraction was an important factor not only for multifidus injury, but also for long-term multifidus atrophy after posterior lumbar surgery; a longer retraction time caused more severe multifidus injury and atrophy. Muscle fibers can be regenerated postoperatively, and denervation might be the reason for muscle atrophy.

Correlation between biomechanical properties of the annulus fibrosus and magnetic resonance imaging (MRI) findings.

PURPOSE: To investigate the regional tensile properties of human annulus fibrosus (AF) and relate them to magnetic resonance imaging (MRI) findings. METHODS: 44 human cadaveric lumbar spines were harvested (24 male, 20 female, aged 25-64 years). MRI was used to identify Pfirrmann grade of disc degeneration, and Modic changes (MCs). Intervertebral discs were then removed and dissected into five regions: nucleus pulposus, anterior AF, anterolateral AF, lateral AF, and posterolateral AF. Samples for tensile testing (1.5 mm × 1.5 mm × 5 mm) were removed from inner, middle and outer parts of each region. RESULTS: 1969 specimens from 189 discs were stretched to failure. Average tensile stiffness (modulus) increased from 4.80 MPa in the inner AF to 13.0 MPa in the outer AF. Strength (UTS) increased similarly, from 1.18 to 3.29 MPa, whereas elongation at failure decreased, from 49 to 38%. The only significant change with age was a reduction in UTS in the middle annulus. In contrast, severe grades of disc degeneration were associated with consistent and highly significant reductions in tensile properties. Effects were greatest in the outer AF, where stiffness and strength fell by 29 and 43%, respectively. Modic changes also were associated with reduced stiffness and strength, but here the effects were greatest in the inner and middle AF. CONCLUSION: Weakening of degenerated AF may be caused by accumulating structural defects, and enzymatic degradation. MRI has the potential to identify local weakening of the AF.

Induction of cervical disc degeneration and discogenic pain by low concentration Propionibacterium acnes infection: an in vivo animal study.

BACKGROUND: Although cervical intervertebral disc (IVD) degeneration is closely associated with neck pain, its cause remains unclear. In this study, an animal model of cervical disc degeneration and discogenic neck pain induced by a low concentration of Propionibacterium acnes (P. acnes-L) is investigated to explore the possible mechanisms of cervical discogenic pain. METHODS: Cervical IVD degeneration and discitis was induced in 8-week-old male rats in C3-C6 IVDs through the anterior intervertebral puncture with intradiscal injections of low and high concentrations of P. acnes (P. acnes-L, n = 20 and P. acnes-H, n = 15) or Staphylococcus aureus (S. aureus, n = 15), compared to control (injection with PBS, n = 20). The structural changes in the cervical IVD using micro-CT, histological evaluation, and gene expression assays after MRI scans at 2 and 6 weeks post-modeling. The P. acnes-L induced IVD degeneration model was assessed for cervical spine MRI, histological degeneration, pain-like behaviors (guarding behavior and forepaw von Frey), nerve fiber growth in the IVD endplate region, and DRG TNF-α and CGRP. RESULTS: IVD injection with P. acnes-L induced IVD degeneration with decreased IVD height and MRI T2 values. IVD injection with P. acnes-H and S. aureus both lead to discitis-like changes on T2-weighted MRI, trabecular bone remodeling on micro-CT, and osseous fusion after damage in the cartilage endplate adjacent to the injected IVD. Eventually, rats in the P. acnes-L group exhibited significant nociceptive hypersensitivity, nerve fiber ingrowth was observed in the IVD endplate region, inflammatory activity in the DRG was significantly increased compared to the control group, and the expression of the pain neurotransmitter CGRP was significantly upregulated. CONCLUSION: P. acnes-L was validated to induce cervical IVD degeneration and discogenic pain phenotype, while P. acnes-H induced was identified to resemble septic discitis comparable to those caused by S. aureus infection.

FATP2 regulates osteoclastogenesis by increasing lipid metabolism and ROS production.

Lipid metabolism plays a crucial role in maintaining bone homeostasis, particularly in osteoclasts (OCs) formation. Here, we found that the expression level of FATP2, a transporter for long-chain and very-long-chain fatty acids, was significantly upregulated during OC differentiation and in the bone marrow of mice fed a high-fat diet (HFD). Notably, the use of FATP2 siRNA or a specific inhibitor (Lipofermata) resulted in significant inhibition of OC differentiation, while only slightly affecting osteoblasts. In pathological models of bone loss induced by LPS or ovariectomy, in vivo treatment with Lipofermata was able to rescue the loss of bone mass by inhibiting OC differentiation. RNA sequencing revealed that Lipofermata reduced fatty acid ß-oxidation and inhibited energy metabolism, while regulating ROS metabolism to decrease ROS production, ultimately inhibiting OC differentiation. Treatment with Lipofermata, either in vivo or in vitro, effectively rescued the overactivation of OCs, indicating that FATP2 regulated OC differentiation by modulating fatty acid uptake and energy metabolism. These findings suggested that targeting FATP2 may represent a promising therapeutic approach for pathological osteoporosis.

The inhibition of osteoclastogenesis by Lipofermata, a FATP2 inhibitor, was achieved through the reprogramming of energy metabolism and regulation of ROS levels. In both pathological bone loss and HFD-induced osteoporosis models, the expression levels of FATP2 were significantly upregulated, and Lipofermata demonstrated potential therapeutic effects in the pathological bone loss model.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches.

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.