Microscopic characteristics of peri- and postmortem fracture surfaces

AUTHORS

Jessica Skinner, Natalie Langley, Samuel Fahrenholtz, Yuktha Shanavas, Brian Waletzki, Robert Brown, James Herrick, Loukham Shyamsunder, Peter Goguen, Subramaniam Rajan

ABSTRACT

This study investigated if microscopic surface features captured with a scanning electron microscope (SEM) effectively discriminate fracture timing. We hypothesized that microscopic fracture characteristics, including delamination, osteon pullout, and microcracks, may vary as bone elasticity decreases, elucidating perimortem and postmortem events more reliably than macroscopic analyses. Thirty-seven unembalmed, defleshed human femoral shafts from males (n=18) and females (n=2) aged 33–81 years were fractured at experimentally simulated postmortem intervals (PMIs) ranging from 1 to 60 warm weather days (250–40,600 ADH). A gravity convection oven was used to approximate tissue decomposition at 37 C and 27 C, and the resulting heat-time unit (accumulated degree hours, or ADH) was used to examine fractures in elastic/wet versus brittle/dry bone. The bones were fractured with a drop test frame using a three-point bending setup, sensors were used to calculate fracture energy, and high-speed photography documented fracture events. The following data were collected to relate fracture appearance to the biomechanical properties of bone: PMI (postmortem interval) length in ADH, temperature, humidity, collagen percentage, water loss, bone mineral density, cortical bone thickness, fracture energy, age, sex, cause of death, and microscopic fracture feature scores. SEM micrographs were collected from the primary tension zones of each fracture surface, and three microscopic fracture characteristics were scored from a region of interest in the center of the tension zone: percentage of delaminated osteons, percent osteon pullout, and number of microcracks. Multiple linear regression showed that microscopic fracture surface features are strong predictors of ADH (adjusted R-squared=0.67 for the 0 – 40,000 ADH samples; adjusted R-squared=0.92 for the 0–16,000 ADH samples). Osteon pullout is the single best predictor of ADH. Additionally, water loss is the primary driver of bone elasticity changes in low ADH samples, while collagen fibers appear to remain intact until later in the postmortem interval (approximately 40,000 ADH in this study). The results of this study indicate microscopic fracture surface analysis detects the biomechanical effects of decreased elasticity more reliably and with greater sensitivity than macroscopic analysis.

NLRP3 blockade by MCC950 suppressed osteoclastogenesis via NF-κB/c-Fos/NFATc1 signal pathway and alleviated bone loss in diabetes mellitus

AUTHORS

Guoping Cai, Xiaoting Song, Hua Luo, Gaoyuan Dai, Honghao Zhang, Dengteng Jiang, Xinhuan Lei, Haixiao Chen, Liwei Zhang

ABSTRACT

Obesity and type 2 diabetes mellitus (T2DM) are linked to osteoporosis development, with obesity being a significant risk factor for T2DM. T2DM patients with obesity exhibit a higher fracture rate and often have a poor prognosis post-fracture. To address the urgent need for understanding the mechanisms of diabetic osteoporosis (DOP), research is ongoing to explore how obesity and T2DM impact bone metabolism. The NLRP3 inflammasome has been implicated in the pathogenesis of osteoporosis, and MCC950, an NLRP3 inflammasome inhibitor, has shown promise in various diseases but its role in osteoporosis remains unexplored. In this study, BMMs and BMSCs were isolated and cultured to investigate the effects of MCC950 on bone metabolism, and DOP model was used to evaluate the efficacy of MCC950 in vivo. The study demonstrated that MCC950 treatment inhibited osteoclast differentiation, reduced bone resorption capacity in BMMs without suppression for osteoblast differentiation from BMSCs. Additionally, MCC950 suppressed the activation of the NF-κB signaling pathway and downregulated key factors associated with osteoclast differentiation. Additionally, MCC950 alleviated bone loss in DOP mouse. These findings suggest that MCC950, by targeting the NLRP3 inflammasome, may have a protective role in preventing osteoporosis induced by T2DM with obesity. The study highlights the potential therapeutic implications of MCC950 in managing diabetic osteoporosis and calls for further research to explore its clinical application in high-risk patient populations.

Bone Marrow Stem Cell Population in Single- and Multiple-Level Aspiration

AUTHORS

Xiangguo Che, Hee-June Kim, Xian Jin, Joon-Woo Kim, Kyeong-Hyeon Park, Jeong-Ok Lim, Hee-Soo Kyung, Chang-Wug Oh, Je-Yong Choi

ABSTRACT

Background: Bone marrow aspiration concentrate (BMAC) has garnered increasing interest due to its potential for healing musculoskeletal injuries. While the iliac crest remains a common harvest site, the aspiration technique’s efficacy in offering the highest yield and prevalence of mesenchymal stem cells (MSCs) is controversial. This study aimed to compare two different techniques of bone marrow aspiration over the anterior iliac crest from a single level versus multiple levels. Methods: Anterior iliac crests were selected in seven adult patients (aged between 31 and 59 years old). Aspiration was achieved using an 11-gauge needle (length: 100 mm; diameter: 2.3 mm) specifically manufactured for bone marrow collection (BD, Becton, Franklin Lakes, NJ, USA) connected to a 10 mL syringe. On one side, 4cc of bone marrow was aspirated at a single level to a depth of 7 cm without changing the needle direction. On the other side, over the same portion of the iliac crest, 1 cc of bone marrow was obtained from multiple levels of different depths during needle retrieval, maintaining a distance of 1 cm and changing the tip direction. The samples were blindly sent to the laboratory without indicating whether they came from an single level or multiple levels. Fluorescence-activated cell sorting (FACS) and osteoblast differentiation were analyzed and compared. Results: In the FACS analysis, the single level resulted in a higher population of MSCs that were positive for CD105, CD73, and CD90 and negative for CD34, compared to the multiple-level method. In the process of osteoblast differentiation, it was observed that MSCs exhibited more advanced features of enhanced osteoblastic abilities in the single-level method rather than the multiple-level method. Conclusions: A single-level aspiration technique at the anterior iliac crest may produce a high-quality bone marrow aspirate. This technique may help obtain specific populations of MSCs with the desired characteristics for use in regenerative therapies for musculoskeletal injuries.

CNSC-12. IMMUNOLOGICALLY TARGETING U1 MUTANT SHH MEDULLOBLASTOMA

AUTHORS

Michelle Kameda , Rong Zheng , Nabil Ahmed , Chonghui Cheng , Michael Taylor

ABSTRACT

OBJECTIVES

Medulloblastoma (MB) is the most common malignant pediatric brain tumor representing a significant burden of morbidity and mortality in the US. MB is comprised of four subgroups: Wnt, Shh, Group 3, and Group 4. Shh tumors represent 25% of cases and subdivides into Shh-beta and Shh-gamma, Shh-alpha, and Shh-delta. Half of Shh MB carry an identical somatic point mutation in a non-coding small nuclear RNA (snRNA) called U1 (r.3A>G) which is found in 97% of Shh-d tumors, and in most Shh-alpha tumors with TP53 mutations. Current therapies for patients with TP53 and U1 mutant Shh-alpha MB observe rare survivors, and adult Shh-delta patients continue to experience significant morbidity and mortality calling for urgent prioritization of these tumors for targeted therapy.

METHODS

Cryptic exons were identified in both Shh-delta U1 snRNA mutant samples and Shh-delta U1 wildtype (WT) samples using CryEx pipeline. In-house scripts were utilized for selecting for cryptic exons that are uniquely expressed in Shh-delta U1 snRNA mutant compared to wildtype (WT) samples.

RESULTS

Analyzing 180 Shh MB RNA-seq samples, we identified 23% Shh-alpha, no Shh-beta, 97% of Shh-delta and 3% Shh-gamma harbored the U1 mutation. The splicing landscape was then interrogated comparing Shh-delta U1 snRNA mutant samples to WT samples. Expressed exons were filtered to exclude known exons to identify novel or cryptic exons. To select for Shh-delta U1 snRNA mutant induced cryptic exons, CryEx arising from introns, not identified in Shh-delta U1 WT and included >10% of their inclusion rates measured by percent spliced in (PSI) in Shh-delta U1 snRNA mutant samples were filtered. Of the middle CryEx, we identified 43,188 that were U1 mutant induced. Further filtering for cell surface Middle CryEx, three of the 75 middle CryEx overlapped.

CONCLUSION

The PTCH1 neoantigen formed from the CryEx insertion translates a protein that is unique to the tumor cells (i.e., not in normal tissue) was identified as a juxtamembrane for therapeutic drug discovery.

Early movement restriction impairs the development of sensorimotor integration, motor skills and memory in rats: Towards a preclinical model of developmental coordination disorder?

AUTHORS

Hanane Khalki, Diego Cabral Lacerda, Corane Karoutchi, Maxime Delcour, Orlane Dupuis, Marine Kochmann, Jean-Michel Brezun, Erwan Dupont, Mamta Amin, Muriel Darnaudéry, Marie-Hélène Canu, Mary F. Barbe, Jacques-Olivier Coq

ABSTRACT

Children with neurodevelopmental disorders, such as developmental coordination disorder (DCD), exhibit gross to fine sensorimotor impairments, reduced physical activity and interactions with the environment and people. This disorder co-exists with cognitive deficits, executive dysfunctions and learning impairments. Previously, we demonstrated in rats that limited amounts and atypical patterns of movements and somatosensory feedback during early movement restriction manifested in adulthood as degraded postural and locomotor abilities, and musculoskeletal histopathology, including muscle atrophy, hyperexcitability within sensorimotor circuitry and maladaptive cortical plasticity, leading to functional disorganization of the primary somatosensory and motor cortices in the absence of cortical histopathology. In this study, we asked how this developmental sensorimotor restriction (SMR) started to impact the integration of multisensory information and the emergence of sensorimotor reflexes in rats. We also questioned the enduring impact of SMR on motor activities, pain and memory. SMR led to deficits in the emergence of swimming and sensorimotor reflexes, the development of pain and altered locomotor patterns and posture with toe-walking, adult motor performance and night spontaneous activity. In addition, SMR induced exploratory hyperactivity, short-term impairments in object-recognition tasks and long-term deficits in object-location tasks. SMR rats displayed minor alterations in histological features of the hippocampus, entorhinal, perirhinal and postrhinal cortices yet no obvious changes in the prefrontal cortex. Taken all together, these results show similarities with the symptoms observed in children with DCD, although further exploration seems required to postulate whether developmental SMR corresponds to a rat model of DCD.

AAV1.tMCK.NT-3 gene therapy improves phenotype in Sh3tc2−/− mouse model of Charcot–Marie–Tooth Type 4C

AUTHORS

Burcak Ozes , Lingying Tong , Kyle Moss , Morgan Myers , Lilye Morrison , Zayed Attia , Zarife Sahenk

ABSTRACT

Charcot–Marie–Tooth Type 4C (CMT4C) is associated with mutations in the SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene, primarily expressed in Schwann cells (SCs). Neurotrophin-3 (NT-3) is an important autocrine factor for SC survival and differentiation, and it stimulates neurite outgrowth and myelination. In this study, scAAV1.tMCK.NT-3 was delivered intramuscularly to 4-week-old Sh3tc2−/− mice, a model for CMT4C, and treatment efficacy was assessed at 6-month post-gene delivery. Efficient transgene production was verified with the detection of NT-3 in serum from the treated cohort. NT-3 gene therapy improved functional and electrophysiological outcomes including rotarod, grip strength and nerve conduction velocity. Qualitative and quantitative histopathological studies showed that hypomyelination of peripheral nerves and denervated status of neuromuscular junctions at lumbrical muscles were also improved in the NT-3-treated mice. Morphometric analysis in mid-sciatic and tibial nerves showed treatment-induced distally prominent regenerative activity in the nerve and an increase in the estimated SC density. This indicates that SC proliferation and differentiation, including the promyelination stage, are normal in the Sh3tc2−/− mice, consistent with the previous findings that Sh3tc2 is not involved in the early stages of myelination. Moreover, in size distribution histograms, the number of myelinated axons within the 3- to 6-µm diameter range increased, suggesting that treatment resulted in continuous radial growth of regenerating axons over time. In conclusion, this study demonstrates the efficacy of AAV1.NT-3 gene therapy in the Sh3tc2−/− mouse model of CMT4C, the most common recessively inherited demyelinating CMT subtype.