Clinical validation of C12FDG as a marker associated with senescence and osteoarthritic phenotypes.

Chronic conditions associated with aging have proven difficult to prevent or treat. Senescence is a cell fate defined by loss of proliferative capacity and the development of a pro-inflammatory senescence-associated secretory phenotype comprised of cytokines/chemokines, proteases, and other factors that promotes age-related diseases. Specifically, an increase in senescent peripheral blood mononuclear cells (PBMCs), including T cells, is associated with conditions like frailty, rheumatoid arthritis, and bone loss. However, it is unknown if the percentage of senescent PBMCs associated with age-associated orthopedic decline could be used for potential diagnostic or prognostic use in orthopedics. Here, we report senescent cell detection using the fluorescent compound C12FDG to quantify PBMCs senescence across a large cohort of healthy and osteoarthritic patients. There is an increase in the percent of circulating C12FDG+ PBMCs that is commensurate with increases in age and senescence-related serum biomarkers. Interestingly, C12FDG+ PBMCs and T cells also were found to be elevated in patients with mild to moderate osteoarthritis, a progressive joint disease that is strongly associated with inflammation. The percent of C12FDG+ PBMCs and age-related serum biomarkers were decreased in a small subgroup of study participants taking the senolytic drug fisetin. These results demonstrate quantifiable measurements in a large group of participants that could create a composite score of healthy aging sensitive enough to detect changes following senolytic therapy and may predict age-related orthopedic decline. Detection of peripheral senescence in PBMCs and subsets using C12FDG may be clinically useful for quantifying cellular senescence and determining how and if it plays a pathological role in osteoarthritic progression.

Evaluating the Morphology of Unique Superficial Fissured Cartilage Lesions at the Femoral Head-Neck Junction in Patients with Femoroacetabular Impingement Syndrome.

Background: While an association between femoroacetabular impingement (FAI) and osteoarthritis (OA) has been reported, the mechanistic differences and transition between the 2 conditions is not fully understood. In FAI, cartilage lesions at the femoral head-neck junction can sometimes be visualized during hip arthroscopy. Purpose/Hypothesis: The purpose of this study was to describe a unique dimpled pattern of superficial fissured cartilage lesions on the femoral head-neck junction at impingement site in patients with FAI syndrome (FAIS) and to evaluate the clinical, histological, and genetic phenotype of this cartilage. We hypothesized that the cartilage lesions may indicate risk for, or predict occurrence of, OA. Study Design: Controlled laboratory study. Methods: Six hips (6 patients; mean age, 34.2 ± 12.9 years; range, 19-54 years) with dimpled or fissured cartilage were included among patients who underwent hip arthroscopy for treatment of FAIS from October 2020 through December 2021. This affected cartilage (dimple-pattern group) and normal cartilage (control group) on the femoral head-neck junction were collected from the same patients and evaluated for histological quantification by Mankin scores and expression of proteins related to cartilage degeneration (eg, matrix metalloproteinase [MMP]-1, MMP-2, MMP-3, MMP-10, and MMP-12, tissue inhibitor of metalloproteinase [TIMP]-1 and TMP-2, aggrecan neopepitope CS846, and hyaluronic acid [HA]) with the use of Milliplex Multiplex Assays. Results: All 6 hips were of the mixed FAI subtype. Preoperatively, 4 of 6 hips had Tönnis grade 1 radiographic changes, which was associated with greater femoral head chondral damage visualized intraoperatively. Mankin scores for the normal cartilage group and the dimple-pattern group were 0.67 ± 0.82 and 3.3 ± 0.82, respectively. Dimple pattern fissured cartilage showed a significant increase in Mankin score (P = .031) and a significant increase in protein expression of CS846 (P = .031) compared with normal cartilage. There were no significant differences in MMPs, TIMPs, or HA levels between the 2 groups. Conclusion: The dimple pattern fissured cartilage, compared to normal cartilage, showed histologically significant cartilage degeneration and a significant increase in protein expression of CS846, a biomarker for early OA. Clinical Relevance: This lesion serves as helpful visual indicator of early degeneration of the cartilage of femoral head-neck junction caused by FAIS.

A Systemic and Local Comparison of Senescence in an Acute Anterior Cruciate Ligament Injury-A Pilot Case Series.

BACKGROUND: Senescence, a characteristic of cellular aging and inflammation, has been linked to the acceleration of osteoarthritis. The purpose of this study is to prospectively identify, measure, and compare senescent profiles in synovial fluid and peripheral blood in patients with an acute knee injury within 48 h. METHODS: Seven subjects, aged 18-60 years, with an acute ACL tear with effusion were prospectively enrolled. Synovial fluid and peripheral blood samples were collected and analyzed by flow cytometry, using senescent markers C12FDG and CD87. The senescent versus pro-regenerative phenotype was probed at a gene and protein level using qRT-PCR and multiplex immunoassays. RESULTS: C12FDG and CD87 positive senescent cells were detected in the synovial fluid and peripheral blood of all patients. Pro-inflammatory IL-1ß gene expression measured in synovial fluid was significantly higher (p = 0.0156) than systemic/blood expression. Senescent-associated factor MMP-3 and regenerative factor TIMP-2 were significantly higher in synovial fluid compared to blood serum. Senescent-associated factor MMP-9 and regenerative factor TGFß-2 were significantly elevated in serum compared to synovial fluid. Correlation analysis revealed that C12FDG++/CD87++ senescent cells in synovial fluid positively correlated with age-related growth-regulated-oncogene (ρ = 1.00, p < 0.001), IFNγ (ρ = 1.00, p < 0.001), IL-8 (ρ = 0.90, p = 0.0374), and gene marker p16 (ρ = 0.83, p = 0.0416). CONCLUSIONS: There is an abundance of senescent cells locally and systemically after an acute ACL tear without a significant difference between those present in peripheral blood compared to synovial fluid. This preliminary data may have a role in identifying strategies to modify the acute environment within the synovial fluid, either at the time of acute ligament injury or reconstruction surgery.

Encapsulation of ß-NGF in injectable microrods for localized delivery accelerates endochondral fracture repair.

Introduction: Currently, there are no non-surgical FDA-approved biological approaches to accelerate fracture repair. Injectable therapies designed to stimulate bone healing represent an exciting alternative to surgically implanted biologics, however, the translation of effective osteoinductive therapies remains challenging due to the need for safe and effective drug delivery. Hydrogel-based microparticle platforms may be a clinically relevant solution to create controlled and localized drug delivery to treat bone fractures. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the shape of microrods, loaded with beta nerve growth factor (ß-NGF) for the purpose of promoting fracture repair. Methods: Herein, PEGDMA microrods were fabricated through photolithography. PEGDMA microrods were loaded with ß-NGF and in vitro release was examined. Subsequently, bioactivity assays were evaluated in vitro using the TF-1 tyrosine receptor kinase A (Trk-A) expressing cell line. Finally, in vivo studies using our well-established murine tibia fracture model were performed and a single injection of the ß-NGF loaded PEGDMA microrods, non-loaded PEGDMA microrods, or soluble ß-NGF was administered to assess the extent of fracture healing using Micro-computed tomography (µCT) and histomorphometry. Results: In vitro release studies showed there is significant retention of protein within the polymer matrix over 168 hours through physiochemical interactions. Bioactivity of protein post-loading was confirmed with the TF-1 cell line. In vivo studies using our murine tibia fracture model show that PEGDMA microrods injected at the site of fracture remained adjacent to the callus for over 7 days. Importantly, a single injection of ß-NGF loaded PEGDMA microrods resulted in improved fracture healing as indicated by a significant increase in the percent bone in the fracture callus, trabecular connective density, and bone mineral density relative to soluble ß-NGF control indicating improved drug retention within the tissue. The concomitant decrease in cartilage fraction supports our prior work showing that ß-NGF promotes endochondral conversion of cartilage to bone to accelerate healing. Discussion: We demonstrate a novel and translational method wherein ß-NGF can be encapsulated within PEGDMA microrods for local delivery and that ß-NGF bioactivity is maintained resulting in improved bone fracture repair.

Wireless Measurements Using Electrical Impedance Spectroscopy to Monitor Fracture Healing.

There is an unmet need for improved, clinically relevant methods to longitudinally quantify bone healing during fracture care. Here we develop a smart bone plate to wirelessly monitor healing utilizing electrical impedance spectroscopy (EIS) to provide real-time data on tissue composition within the fracture callus. To validate our technology, we created a 1-mm rabbit tibial defect and fixed the bone with a standard veterinary plate modified with a custom-designed housing that included two impedance sensors capable of wireless transmission. Impedance magnitude and phase measurements were transmitted every 48 h for up to 10 weeks. Bone healing was assessed by X-ray, µCT, and histology. Our results indicated the sensors successfully incorporated into the fracture callus and did not impede repair. Electrical impedance, resistance, and reactance increased steadily from weeks 3 to 7-corresponding to the transition from hematoma to cartilage to bone within the fracture gap-then plateaued as the bone began to consolidate. These three electrical readings significantly correlated with traditional measurements of bone healing and successfully distinguished between union and not-healed fractures, with the strongest relationship found with impedance magnitude. These results suggest that our EIS smart bone plate can provide continuous and highly sensitive quantitative tissue measurements throughout the course of fracture healing to better guide personalized clinical care.

Lateral semi-circular canal asymmetry in females with idiopathic scoliosis.

PURPOSE: Adolescent idiopathic scoliosis (AIS) is a three-dimensional spinal structural deformity that occurs in otherwise normal individuals. Although curve progression and severity vary amongst individuals, AIS can lead to significant cosmetic and functional deformity. AIS etiology has been determined to be genetic, however, exact genetic and biological processes underlying this disorder remain unknown. Vestibular structure and function have potentially been related to the etiopathogenesis of AIS. Here, we aimed to characterize the anatomy of the semicircular canals (SCC) within the vestibular system through a novel approach utilizing T2-weighted magnetic resonance images (MRI). METHODS: Three dimensional, MRI-based models of the SCCs were generated from AIS subjects (n = 20) and healthy control subjects (n = 19). Linear mixed models were used to compare SCC morphological measurements in the two groups. We compared side-to-side differences in the SCC measurements between groups (group*side interaction). RESULTS: Side-to-side differences in the lateral SCC were different between the two groups [false discovery rate adjusted p-value: 0.0107]. Orientation of right versus left lateral SCC was significantly different in the AIS group compared to the control group [mean side-to-side difference: -4.1°, 95% CI: -6.4° to -1.7°]. Overall, among subjects in the AIS group, the left lateral SCC tended to be oriented in a more horizontal position than subjects in the control group. SIGNIFICANCE: Asymmetry within the SCCs of the vestibular system of individuals with AIS potentially results in abnormal efferent activity to postural muscles. Consequences of this muscular activity during periods of rapid growth, which often coincides with AIS onset and progression, warrant consideration.

Polyacrylamide Hydrogels Produce Hydrogen Peroxide from Osmotic Swelling in Aqueous Media.

This work demonstrates that hydrogen peroxide (H2O2) is generated in weak polyacrylamide hydrogels due to mechanochemical reactions to osmotic swelling. Hydrogels are important tools and materials for many biomedical applications, particularly for growth of stem cells. However, swollen gels are under constant tension, which makes their individual chains susceptible to mechanochemical bond breakage. In this work, an assay was developed to measure the generation of H2O2 as a result of hydrogel swelling. Polyacrylamide hydrogels with both weak disulfide and strong PEG-diacrylate crosslinkers were synthesized and swelled. H2O2 generation increased in the presence of weaker crosslinkers, up to 30 µM H2O2, whereas stronger crosslinkers reduced this to 5 µM H2O2. H2O2 levels decreased when swelled in the presence of dextran to reduce osmotic stress or increased if the gels were conjugated to an acrylated surface. Finally, H2O2 continued to form for days after the gels had reached their equilibrium sizes, independently of dissolved oxygen. The results of this work impact those working in the 3D cell culture community and demonstrate that even well-characterized systems undergo mechanochemical processes in mild environments.