Excessive R-loops trigger an inflammatory cascade leading to increased HSPC production.

Hematopoietic stem and progenitor cells (HSPCs) arise during embryonic development and are essential for sustaining the blood and immune systems throughout life. Tight regulation of HSPC numbers is critical for hematopoietic homeostasis. Here, we identified DEAD-box helicase 41 (Ddx41) as a gatekeeper of HSPC production. Using zebrafish ddx41 mutants, we unveiled a critical role for this helicase in regulating HSPC production at the endothelial-to-hematopoietic transition. We determined that Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular fitness. Excess R-loop levels in ddx41 mutants triggered the cGAS-STING inflammatory pathway leading to increased numbers of hemogenic endothelium and HSPCs. Elevated R-loop accumulation and inflammatory signaling were observed in human cells with decreased DDX41, suggesting possible conservation of mechanism. These findings delineate that precise regulation of R-loop levels during development is critical for limiting cGAS-STING activity and HSPC numbers.

Bilateral Continuous Automated Distraction Osteogenesis: Proof of Principle.

The purpose of this study was to demonstrate that automated, continuous, curvilinear distraction osteogenesis (DO) in a minipig model is effective when performed bilaterally, at rates up to 3 mm/day, to achieve clinically relevant lengthening. A Yucatan minipig in the mixed dentition phase underwent bilaterally, at a continuous DO at a rate of 2 mm/day at the center of rotation; 1.0 and 3.0 mm/day at the superior and inferior regions, respectively. The distraction period was 13 days with no latency period. Vector and rate of distraction were remotely monitored without radiographs, using the device sensor. After fixation and euthanasia, the mandible and digastric muscles were harvested. The ex vivo appearance, stability, and radiodensity of the regenerate were evaluated using a semiquantitative scale. Percent surface area (PSA) occupied by bone, fibrous tissue, cartilage, and hematoma were calculated using histomorphometrics. The effects of DO on the digastric muscles and mandibular condyles were assessed via microscopy, and degenerative changes were quantified. The animal was distracted to 21 mm and 24 mm on the right and left sides, respectively. Clinical appearance, stability, and radiodensity were scored as "3" bilaterally indicating osseous union. The total PSA occupied by bone (right = 75.53 ±â€Š2.19%; left PSA = 73.11 ±â€Š2.18%) approached that of an unoperated mandible (84.67 ±â€Š0.86%). Digastric muscles and condyles showed negligible degenerative or abnormal histologic changes. This proof of principle study is the first report of osseous healing with no ill-effect on associated soft tissue and the mandibular condyle using bilateral, automated, continuous, and curvilinear DO at rates up to 3 mm/day. The model approximates potential human application of continuous automated distraction with a semiburied device.

Assessment of the OsteoMark-Navigation System for Oral and Maxillofacial Surgery.

PURPOSE: To assess the accuracy of a novel navigation system for maxillofacial surgery using human cadavers and a live minipig model. MATERIALS AND METHODS: We tested an electromagnetic tracking system (OsteoMark-Navigation) that uses simple sensors to determine the position and orientation of a hand-held pencil-like marking device. The device can translate 3-dimensional computed tomographic data intraoperatively to allow the surgeon to localize and draw a proposed osteotomy or the resection margins of a tumor on bone. The accuracy of the OsteoMark-Navigation system in locating and marking osteotomies and screw positions in human cadaver heads was assessed. In group 1 (n = 3, 6 sides), OsteoMark-Navigation marked osteotomies and screw positions were compared to virtual treatment plans. In group 2 (n = 3, 6 sides), marked osteotomies and screw positions for distraction osteogenesis devices were compared with those performed using fabricated guide stents. Three metrics were used to document the precision and accuracy. In group 3 (n = 1), the system was tested in a standard operating room environment. RESULTS: For group 1, the mean error between the points was 0.7 mm (horizontal) and 1.7 mm (vertical). Compared with the posterior and inferior mandibular border, the mean error was 1.2 and 1.7 mm, respectively. For group 2, the mean discrepancy between the points marked using the OsteoMark-Navigation system and the surgical guides was 1.9 mm (range 0 to 4.1). The system maintained accuracy on a live minipig in a standard operating room environment. CONCLUSION: Based on this research OsteoMark-Navigation is a potentially powerful tool for clinical use in maxillofacial surgery. It has accuracy and precision comparable to that of existing clinical applications.

Skeletal and soft tissue response to automated, continuous, curvilinear distraction osteogenesis.

PURPOSE: To document the bone formation and soft tissue changes in response to automated, continuous, curvilinear distraction osteogenesis (DO) at rates greater than 1 mm/day in a minipig model. MATERIALS AND METHODS: Two groups of Yucatan minipigs underwent automated, continuous, curvilinear DO of the right mandible: group A, 1.5 mm/day (n = 5); and group B, 3.0 mm/day (n = 5). Each minipig underwent 12 mm of distraction followed by 24 days of fixation. The distracted and contralateral mandibles were harvested at the end of fixation. The percentage of surface area (PSA) of the regenerate occupied by bone, fibrous tissue, cartilage, and hematoma was determined using computerized histomorphometric analysis. The control groups consisted of DO wounds distracted discontinuously at 1 mm/day and the nonoperated contralateral mandible. The ipsilateral and contralateral digastric muscles were harvested and stained for proliferating cell nuclear antigen (PCNA), myogenic differentiation-1 (MyoD), and paired Box 7 protein (PAX7). RESULTS: All 10 minipigs completed the distraction and fixation period. The PSA occupied by bone was similar for groups A (PSA 64.36% ± 5.87%) and B (PSA 63.83% ± 3.37%) and the control group (1 mm/day; PSA 64.89% ± 0.56%) but was less than that on the nonoperated side (PSA 84.67% ± 0.86%). The PSA occupied by cartilage and hematoma in all groups was minimal (<1.1%). The digastric muscles had no abnormal tissue or inflammation, and PAX7, MyoD, and PCNA expression had returned to the baseline levels. CONCLUSIONS: The results of the present study indicate that bone formation in response to automated, continuous, and curvilinear DO at a rate of 1.5 and 3.0 mm/day is nearly identical to that with discontinuous DO at 1 mm/day. In addition, no deleterious effects were found on the digastric muscles.

Automated continuous distraction osteogenesis may allow faster distraction rates: a preliminary study.

PURPOSE: To determine if automated continuous distraction osteogenesis (DO) at rates faster than 1 mm/day results in bone formation by clinical and radiographic criteria, in a minipig model. MATERIALS AND METHODS: An automated, continuous, curvilinear distraction device was placed across a mandibular osteotomy in 10 minipigs. After 12 mm of distraction and 24 days of fixation, the animals were sacrificed and bone healing was evaluated. The continuous distraction rates were 1.5 mm/day (n = 5) and 3 mm/day (n = 5). A semiquantitative scale was used to assess the ex vivo clinical appearance of the distraction gap (3 = osteotomy not visible; 2 = <50% visible; 1 = >50% visible; 0 = 100% visible), stability (3 = no mobility; 2 and 1 = mobility in 1 plane or 2 planes, respectively; 0 = mobility in 3 planes), and radiographic density (4 = 100% of gap opaque; 3 = >75%; 2 = 50% to 75%; 1 = <50%; 0 = radiolucent). Groups of 4 minipigs distracted discontinuously at 1, 2, and 4 mm/day served as controls. RESULTS: Automated, continuous DO at 1.5-mm/day and 3-mm/day had similar bone formation compared to discontinuous DO at 1-mm/day. The continuous DO 1.5-mm/day group had significantly higher scores for appearance and radiographic density compared with the discontinuous 4-mm/day group. The continuous DO 3-mm/day group had significantly higher scores for appearance and radiographic density compared with the discontinuous 4-mm/day group and greater stability compared with the discontinuous 2- and 4-mm/day groups. CONCLUSIONS: Results of this preliminary study indicate that continuous DO at rates of 1.5 and 3.0 mm/day produces better bone formation compared with discontinuous DO at rates faster than 1 mm/day.