Advancing skeletal health and disease research with single-cell RNA sequencing.

Orthopedic conditions have emerged as global health concerns, impacting approximately 1.7 billion individuals worldwide. However, the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders. The advent of single-cell RNA sequencing (scRNA-seq) technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity. Nevertheless, investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges. In this comprehensive review, we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines. By utilizing these methodologies, crucial insights into the developmental dynamics, maintenance of homeostasis, and pathological processes involved in spine, joint, bone, muscle, and tendon disorders have been uncovered. Specifically focusing on the joint diseases of degenerative disc disease, osteoarthritis, and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension. These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.

Spatially multicellular variability of intervertebral disc degeneration by comparative single-cell analysis.

Previous studies have revealed cellular heterogeneity in intervertebral discs (IVDs). However, the cellular and molecular alteration patterns of cell populations during degenerative progression remain to be fully elucidated. To illustrate the cellular and molecular alteration of cell populations in intervertebral disc degeneration (IDD), we perform single cell RNA sequencing on cells from four anatomic sites of healthy and degenerative goat IVDs. EGLN3+ StressCs, TGFBR3+ HomCs and GPRC5A+ RegCs exhibit the characteristics associated with resistance to stress, maintaining homeostasis and repairing, respectively. The frequencies and signatures of these cell clusters fluctuate with IDD. Notably, the chondrogenic differentiation programme of PROCR+ progenitor cells is altered by IDD, while notochord cells turn to stemness exhaustion. In addition, we characterise CAV1+ endothelial cells that communicate with chondrocytes through multiple signalling pathways in degenerative IVDs. Our comprehensive analysis identifies the variability of key cell clusters and critical regulatory networks responding to IDD, which will facilitate in-depth investigation of therapeutic strategies for IDD.

The Host CYP1A1-Microbiota Metabolic Axis Promotes Gut Barrier Disruption in Methicillin-Resistant Staphylococcus aureus-Induced Abdominal Sepsis.

Background: Host-microbiota crosstalk has been implicated in multiple host metabolic pathway axes that regulate intestinal barrier function. Although constitutive cytochrome P4501A1 (CYP1A1) expression perturbs the microbiome-derived autoregulatory loop following enteric infection, little is known about the role of host CYP1A1 in modulating gut microbiome-mediated signaling during methicillin-resistant Staphylococcus aureus (MRSA)-induced abdominal sepsis and its effects on intestinal barrier integrity. Methods: Abdominal sepsis was induced by the intraperitoneal injection of MRSA in mice. The effect of CYP1A1 deficiency on gut barrier integrity was investigated using RNA sequencing, microbiome analyses, and targeted metabolomics. The microbiota-produced metabolites were validated in patients with sepsis and persistent MRSA infection. Results: Mice lacking CYP1A1 exhibited an altered gut microbiome, a reduced metabolic shift from lysine to cadaverine in the caecal contents and antimicrobial molecule production (Retnlb, Gbp7, and Gbp3), and they were protected against gut barrier disruption when subjected to MRSA challenge. These beneficial effects were validated in aryl hydrocarbon receptor (AHR) knockout (KO) mice by cohousing with CYP1A1 KO mice and abrogated after supplementation with cadaverine or Enterococcus faecalis, the primary microbiota genus for cadaverine synthesis. Antibiotic-driven gut dysbacteriosis impaired the survival benefit and disrupted the intestinal barrier integrity in CYP1A1 KO mice after MRSA infection. Furthermore, increased cadaverine levels in feces and serum were detected in critically ill patients with gut leakiness during persistent MRSA infection, whereas cadaverine was not detected in healthy controls. Additionally, microbiota-derived cadaverine induced enterocyte junction disruption by activating the histamine H4 receptor/nuclear factor-κB/myosin light-chain kinase signaling pathway. Conclusion: This study revealed the unexpected function of host CYP1A1 in microbiota-mediated cadaverine metabolism, with crucial consequences for dysbacteriosis following MRSA-induced abdominal sepsis, indicating that inhibiting CYP1A1 or blocking cadaverine-histamine H4 receptor signaling could be a potential therapeutic target against abdominal sepsis. Clinical Trial Registration: [http://www.chictr.org.cn/index.aspx], identifier [ChiCTR1800018646].

Outcomes in Thoracolumbar and Lumbar Traumatic Fractures: Does Restoration of Unfused Segmental Mobility Correlated to Implant Removal Time?

BACKGROUND: Posterior fixation without fusion can treat thoracolumbar and lumbar traumatic fractures effectively in certain cases. However, whether patients benefit from implant removal and the correlation between the range of motion (ROM) of the involved segments and the removal time have not been determined. METHODS: From 2018 to 2020, we retrospectively reviewed data of patients with AO spine type A or B thoracolumbar or lumbar traumatic fractures who underwent implant removal. A total of 17 patients (group A), 21 patients (group B), and 12 patients (group C) underwent implant removal after the index surgery within 12 months, between 12 and 24 months, and over 24 months, respectively. Clinical and radiological outcomes, including visual analog scale for back pain, patient satisfaction, Oswestry disability index, and EuroQol 5 dimensions questionnaire, for quality of life and segmental ROM were analyzed. RESULTS: The average follow-up time was 9.1 ± 5.7 months after implant removal. There were no significant differences in visual analog scale and patient satisfaction among the 3 groups at the same observation time point. Among the 3 groups, patients in group A gained the lowest Oswestry disability index and highest EuroQol 5 dimensions questionnaire scores after removal and at the final follow-up. The best ROM was obtained in group A followed by groups B and C (11.5° ± 6.2°, 5.5° ± 1.6°, and 2.4° ± 0.6°, respectively). CONCLUSIONS: Immobilization of the involved segments over 24 months may lead to loss of ROM. Regained segmental ROM is correlated negatively with implant removal time, and removal within 12 months promises a better ROM and quality of life.

Intraarticular bone grafting in atlantoaxial facet joints via a posterior approach: nonstructural or structural-a minimum 24-month follow-up.

OBJECTIVE: To investigate the necessity of nonstructural or structural intraarticular bone grafting in atlantoaxial facet joints via a posterior approach and the influence by the presence of basilar invagination (BI). METHODS: From November 2016 to October 2018, patients who underwent posterior atlantoaxial or occipitocervical arthrodesis surgery at one institute were retrospectively reviewed. Operation records, preoperative and postoperative clinical status, and radiological films were analyzed. RESULTS: Thirty-three patients (19 without BI, 14 with BI) underwent posterior facet joint release followed by intraarticular bone grafting were enrolled finally. Twenty-four nonstructural (15 without BI, 9 with BI) and 9 structural (4 without BI, 5 with BI) grafting were performed. The average follow-up was 32.15±6.73 months (24-47 months). Among them, 1 (3.03%) implant failure occurred, and 32 (96.97%) achieved satisfactory neurological outcomes, including 28 (84.85%) complete and 4 (12.12%) acceptable reductions with complete fusion within 6 months. For patients without BI, structural and nonstructural grafting showed no significant difference in terms of reduction maintenance (100% vs 73.33%, p = 0.530), while for those with BI, structural grafting significantly increased the postoperative height of the joint space (5.67±1.22 mm vs 3.43±1.78 mm, p = 0.002) and maintained it much better than nonstructural grafting (88.89% vs 20.00%, p = 0.023), contributing notably to BI correction. CONCLUSION: Intraarticular structural bone grafting in atlantoaxial facet joints has the advantage of maintaining anterior column height in the case of lateral mass collapse or when BI correction is needed; otherwise, nonstructural bone grafting is enough.

Comparison of unilateral and bilateral polymethylmethacrylate-augmented cannulated pedicle screw fixation for the management of lumbar spondylolisthesis with osteoporosis.

BACKGROUND: Cannulated pedicle screw (CPS) augmented by polymethylmethacrylate (PMMA) can achieve satisfactory clinical efficacy in the treatment of lumbar spondylolisthesis with osteoporosis. However, accurate application of CPSs will help to avoid the difficulty of screw revision and reduce the incidence of PMMA-related complications. This study aimed to investigate the mid-term efficacy of CPS compared to unilateral and bilateral applications in this common lumbar degenerative disease. METHODS: May 2011 and May 2018, 50 patients with lumbar spondylolisthesis with osteoporosis who underwent posterior fixation and fusion using traditional pedicle screws or CPSs were included in the study. Patients were divided into two groups based on the application: the unilateral PMMA-augmented CPS group (UC, n = 29) and the bilateral PMMA-augmented CPS group (BC, n = 21). Operation time, blood loss, average hospitalization time, PMMA leakage, and other complications were recorded. The visual analog scale (VAS) and Oswestry disability index (ODI) scores were used to evaluate symptom recovery. Radiographic results were compared for intervertebral fusion and screw loosening. RESULTS: There were no significant differences in the baseline data of the two groups. The VAS and ODI scores improved significantly after surgery (P < 0.05), with no significant differences between the groups (P > 0.05). The operation time and blood loss in the UC group were significantly lower than those in the BC group (P < 0.05). However, the loss of intervertebral disk height and Taillard index did not differ significantly between the groups. The rates of PMMA leakage in the UC and BC groups were 7.0% and 11.9%, respectively (P < 0.05). Bony fusion was achieved in all groups without screw loosening at the last follow-up. Only one patient experienced superficial infection in both groups, while cerebrospinal fluid leakage was observed in two patients in the BC group. CONCLUSIONS: Unilateral application of PMMA-augmented CPS may provide adequate clinical safety and effectiveness in the surgical treatment of lumbar spondylolisthesis with osteoporosis.

Posterolateral fusion combined with posterior decompression shows superiority in the treatment of severe lumbar spinal stenosis without lumbar disc protrusion or prolapse: a retrospective cohort study.

BACKGROUND: Currently, discectomy and posterior decompression combined with lumbar circumferential fusion (CF) have been accepted as a major procedure for severe lumbar spinal stenosis (LSS). However, studies on severe LSS without protruded intervertebral disc to minimize study bias are lacking. We aimed to investigate the effectiveness of sole posterior decompression with lumbar posterolateral fusion (PLF) and the necessity of discectomy and CF in patients with severe LSS without lumbar disc protrusion or prolapse. METHODS: This retrospective cohort study included 153 severe LSS patients without lumbar disc protrusion or prolapse who were admitted in a tertiary spine center with at least a 2-year follow-up between January 2014 and August 2017. Patients were divided into the PLF (n = 77; those who underwent posterior decompression with PLF in 1-3 segments) or CF (n = 76; those who underwent posterior decompression and discectomy with CF in 1-3 segments) groups. Pedicle screw instrumentation was applied to avoid postoperative instability. Clinical outcomes were assessed by visual analog scale (VAS), Oswestry Disability Index (ODI), and Japanese Orthopedic Association Score (JOA, lumbar pain score). Duration of operation, blood loss, surgical cost, and postoperative complications were analyzed. Height of intervertebral space, lumbar lordosis, and bone union were confirmed by lumbar radiography or computed tomography. RESULTS: Both groups achieved significant improvement in JOA, ODI, and VAS compared with preoperative values (P < 0.001), but without significant difference between the two groups. Both groups achieved high fusion rate without difference and correction of lumbar lordosis and intervertebral space height (P < 0.001), especially in the CF group (P < 0.05). Duration of operation, blood loss, and operation cost were significantly higher in the CF group than in the PLF group (P < 0.001). Eight complications were found in both groups (1, PLF group; 7, CF group; P < 0.05). CONCLUSIONS: After posterior decompression, PLF successfully achieves bony fusion and symptom relief with lower complication rate, lesser surgical blood loss, shorter operative time, and lesser cost than CF. Thus, sole posterior decompression with PLF is an effective treatment for severe LSS without lumbar disc protrusion or prolapse.

Back Pain-Inducing Test, a Novel and Sensitive Screening Test for Painful Osteoporotic Vertebral Fractures: A Prospective Clinical Study.

To detect painful vertebral fractures (VFs) in back pain populations at risk of osteoporosis, we designed a physical examination test (the Back Pain-Inducing Test [BPIT]) that included three movements: lying supine, rolling over, and sitting up. If back pain is induced during any of these movements, the result is defined as positive, thereby establishing a presumptive diagnosis of painful VFs. Pain severity is quantified using a self-reported numerical rating scale (NRS). The presence or absence of painful VFs is verified by whole-spine magnetic resonance imaging (MRI), the gold standard for final diagnosis. According to the standards for reporting diagnostic accuracy, a real-world, prospective, and observational study was performed on 510 back pain patients (enrolled from a single institute) at risk of osteoporosis. The sensitivity, specificity, and accuracy of the BPIT for identifying painful VFs were 99.1% (95% CI, 97.5% to 99.8%), 67.9% (95% CI, 60.4% to 74.5%), and 89.0%, respectively. The positive and negative predictive values were 86.6% (95% CI, 82.9% to 89.6%) and 97.4% (95% CI, 92.6% to 99.3%), respectively. Cutoff NRS scores for lying supine, rolling over, and sitting up were 3, 0, and 2, respectively. The corresponding area under the receiver operating characteristic curves (AUROCs) of each movement was 0.898 (95% CI, 0.868 to 0.922), 0.884 (95% CI, 0.854 to 0.911), and 0.910 (95% CI, 0.882 to 0.933), respectively. Although the high prevalence of VFs in the enrolled cohort partially limits the external validity of the predictive value in the general population, we conclude that the BPIT is potentially effective for detecting painful VFs in back pain populations at risk of osteoporosis. This test may be used as a stratification tool in decision-making on subsequent imaging procedures: a negative BPIT rules out painful VFs and indicates that an MRI should be spared, whereas a positive BPIT means that an MRI is necessary and is likely to identify painful VFs. © 2019 American Society for Bone and Mineral Research.