Cell lineage specification during development of the anterior lateral plate mesoderm and forelimb field

The lateral plate mesoderm (LPM) is a transient embryonic tissue that gives rise to a diverse range of mature cell types, including the cardiovascular system, the urogenital system, endoskeleton of the limbs, and mesenchyme of the gut. While the genetic processes that drive development of these tissues are well defined, the early cell fate choices underlying LPM development and specification are poorly understood. In this study, we utilize single-cell transcriptomics to define cell lineage specification during development of the anterior LPM and the forelimb field in the chicken embryo. We identify the molecular pathways directing differentiation of the aLPM towards a somatic or splanchnic cell fate, and subsequent emergence of the forelimb mesenchyme. We establish the first transcriptional atlas of progenitor, transitional and mature cell types throughout the early forelimb field and uncover the global signalling pathways which are active during LPM differentiation and forelimb initiation. Specification of the somatic and splanchnic LPM from undifferentiated mesoderm utilizes distinct signalling pathways and involves shared repression of early mesodermal markers, followed by activation of lineage-specific gene modules. We identify rapid activation of the transcription factor TWIST1 in the somatic LPM preceding activation of known limb initiation genes, such as TBX5, which plays a likely role in epithelial-to-mesenchyme transition of the limb bud mesenchyme. Furthermore, development of the somatic LPM and limb is dependent on ectodermal BMP signalling, where BMP antagonism reduces expression of key somatic LPM and limb genes to inhibit formation of the limb bud mesenchyme. Together, these findings provide new insights into molecular mechanisms that drive fate cell choices during specification of the aLPM and forelimb initiation.

Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells

ABSTRACTHuman pluripotent stem cell (hPSC)-induced NK (iNK) cells are a promising “off-the-shelf” cell product for universal immune therapy. Conventional methods for iNK cell regeneration from hPSCs include embryonic body-formation and feeder-based expansion steps, which bring instability, time-consuming, and high costs for manufacture. In this study, we develop an embryonic body-free, organoid aggregate method for NK cell regeneration from hPSCs. In a short time window of 27-day induction, millions of hPSC input can produce over billions of iNK cells without the necessity of NK cell-expansion feeders. The iNK cells highly express classical toxic granule proteins, apoptosis-inducing ligands, as well as abundant activating and inhibitory receptors. Functionally, the iNK cells eradicate human tumor cells by mechanisms of direct cytotoxity, apoptosis, and antibody-dependent cellular cytotoxicity. This study provides a reliable scale-up method for regenerating human NK cells from hPSCs, which promotes the universal availability of NK cell products for immune therapy.

Biomechanical Evaluation of Oblique Lateral Locking Plate System for Oblique Lumbar Interbody Fusion: A Finite Element Analysis

Objective: Oblique lateral locking plate system (OLLPS) with the locking and reverse pedicle track screw configuration is a novel internal fixation designed for oblique lumbar interbody fusion(OLIF). It is placed in a single-position through the oblique lateral surgical corridor to reduce operative time and subsequent complications of prolonged anesthesia and prone positioning. The purpose of this study was to verify the biomechanical effect of OLLPS.Methods: The intact finite element model of L1–S1（Intact） was established based on CT images of a healthy male volunteer. The L4-L5 intervertebral space was selected as the surgical segment. The surgical models were established separately according to the OLIF surgical procedures and the different internal fixations: (1) stand-alone OLIF (SA); (2) OLIF with 2-screw lateral plate (LP-2); (3) OLIF with 4-screw lateral plate (LP-4); (4) OLIF with OLLPS (OLLPS); and (5) OLIF with bilateral pedicle screw fixation (BPS). After validating the intact model, the physiological loading was applied to the superior surface of L1 to simulate flexion, extension, left bending, right bending, left rotation, and right rotation motions. The evaluation indexes included the L4/5 range of motion (ROM), the L4 maximum displacement, and the maximum stress of the superior and inferior endplate, cage, and supplemental fixation.Results: In OLIF surgery, OLLPS provided multiplanar stability which was similar to that of BPS. Compared with LP-2 and LP-4, OLLPS had the better biomechanical properties in enhancing the instant stability of the surgical segment, reducing the stress of the superior and inferior endplates of the surgical segment, and reducing the risk of cage subsidence.Conclsions: With the minimally invasive background, OLLPS can be an alternative to BPS in OLIF and has a better prospect of clinical promotion and application.

Radiological Assessment of Hip Fracture Union RUSH and Modified RUSH

Objective：Intertrochanteric fracture is a common senile disease, which is mainly treated by surgery. The evaluation of postoperative healing of such fractures has always been based on qualitative evaluation of clinical and radiological indicators. Currently, there are no quantitative evaluations of hip fracture union other than the Radiographic Union Score or Hip (RUSH) score. The aim of this study was to evaluate fracture healing of intertrochanteric fractures treated with intramedullary nailing and plates using RUSH and the modified RUSH score we developed. Methods：We collected a total of 96 patients with surgically treated intertrochanteric fractures, including 46 with lateral plate fixation and 50 with intramedullary nailing. Six orthopedic surgeons assessed the overall impression of fracture union on more than 200 postoperative X-rays without knowing any information, followed by the use of RUSH and modified RUSH to evaluate the x-rays separately to see if the two methods improved the consistency of intertrochanteric fracture union. Results：the consistency of overall fracture healing impression was moderate（ICC=0.487）, RUSH and modified RUSH improved the consistency to substantial（ICC=0.80）and basically perfect（ICC=0.81）, respectively. In addition, for the evaluation of union of all intertrochanteric fractures, the overall score of plate was always higher than that of intramedullary nail. When the lateral plate and intramedullary nail were separately scored, the RUSH score of plate was basically the same as modified RUSH, while the modified RUSH of intramedullary nail was higher than modified RUSH. Ultimately,the medial (r=0.54 and R =0.53) and anterior (r=0.55 and R =0.54) and the global score (R =0.68 and R =0.68) in the single cortex showed a high correlation with the overall fracture healing impression in both RUSH and modified RUSH scores. Ultimately,medial (r=0.54 and r =0.53) and anterior (r=0.55 and r=0.54) in individual cortices and overall scores (r=0.68 and r =0.68) showed a high correlation with overall fracture healing impressions in both RUSH and modified RUSH scores. Conclusions：we developed a new hip fracture score comparable to the RUSH score that significantly improves the consistency of radiographic assessment of intertrochanteric fracture union. Most importantly, modified RUSH filled the callus formation gap in RUSH scores between cortical bridging and no cortical bridging. Therefore, we recommend the use of RUSH or a modified RUSH score to improve orthopedic surgeons' assessment of intertrochanteric fracture union.

Oblique Lateral Interbody Fusion Combined with Lateral Plate VS. Combined with Posterior Pedicle Screw Fixation in Lumbar Degenerative Diseases

Background The oblique lateral interbody fusion (OLIF) is a minimally invasive indirect decompression technique for the treatment of degenerative spinal disease. OLIF with posterior pedicle screws fixation frequently is performed, whereas it requires much more surgery time and blood loss. The purpose of this study was to compare the oblique lateral interbody fusion (OLIF) combined with lateral plate (LP) vs. OLIF combined with posterior pedicle screw (PS) fixation for the treatment of lumbar degenerative diseases.Methods The clinical data of 53 patients with lumbar degenerative diseases who underwent OLIF from January 2020 to September 2020 were retrospectively analyzed，24 in OLIF combined with lateral plate (OLIF+LP) group and 29 in OLIF combined with pedicle screw (OLIF+PS) group. All patients completed a minimum 1-year follow-up. The duration of operation, blood loss, fusion rate and complications were recorded. The visual analog scale (VAS) score, Oswestry Disability Index (ODI), disc height (DH), foraminal height (FH) and cross-sectional area (CSA) were also evaluated.Results The operation time was 75.41±11.53 min in the OLIF+LP group, which shorter than that in OLIF+PS group(127.05±5.62min, P<0.01). Also, the blood loss was significantly less in the OLIF+LP group (39.55±5.32 ml ) than in the OLIF+PS group (89.81±9.62, P<0.01). The VAS and ODI scores both significantly reduced after operation in OLIF+LP group. There was no difference either in VAS or ODI scores by 1 year after surgery between two groups (P>0.01). The DH, FH, and CSA parameters were all improved significantly after operation in both groups, however, there was no significant difference at the any follow-up point between the two groups. The total complication rate was 13.21% (7/53) in this study, and there was no siginificant difference between the two groups. The fusion rate was 91.67% in the OLIF+LP group and 93.10% in the OLIF+PS group (P =0.69).Conclusions OLIF+LP fixation seems to be a valuable surgical option for single-segmental lumbar degenerative disease, it can achieve much better clinical outcomes than OLIF+PS group.

Double-layered two-directional somatopleural cell migration during chicken body wall development revealed with local fluorescent tissue labeling

The ventral body wall is derived from the somatic layer of the lateral plate mesoderm, somatopleure, and somite. The primary ventral body wall is formed as a result of the lateral growth of the somatopleure. The secondary body wall is generated through the migration of somitic cells into the somatopleure. While it is reported that the cervical somatopleural cells migrate caudally to the thoracic region during body wall development, the migration of the thoracic somatopleural cells has not been elucidated. To investigate the migration behavior of the somatopleural cells in the thorax during chicken ventral body wall development, we labeled the thoracic somatopleural cells of one somite wide by DiI labeling or gene transfection of enhanced green fluorescent protein and observed the three-dimensional distribution of the labeled cells with the tissue-clearing technique FRUIT. Our labeling experiments revealed the rostral migration of the somatopleural cells into a deep part of the thoracic body wall in embryonic day 6.5 chickens. For embryonic day 8.5 chickens, these deep migrating somatopleural cells were found around the sternal ribs. Thus, we identified the double-layered two-directional migrating pathways of the somatopleural cells: the rostral migration of the deep somatopleural cells and the lateral migration of the superficial somatopleural cells. Our findings imply that the rostral migration of deep somatopleural cells and the lateral migration of superficial ones might be associated with the developing sternal ribs and the innervation of the thoracic cutaneous nerves, respectively.

Clavicular osteotomy to assess subclavian vein injury during internal fixation of midshaft clavicle fractures

Midshaft clavicle fractures are commonly fixed with locking plates. The subclavian vein risks injury during this procedure and the consequences can be fatal. The purpose of this present study is to describe a clavicular osteotomy technique in order to equip orthopaedic surgeons with a means of rapidly accessing a subclavian vein injury. The osteotomy should only be performed following an urgent intraoperative vascular surgery assessment. There must be shared consensus from both orthopaedic and vascular surgery that direct repair of the subclavian vein is necessary, and further exposure is required. The results of the technique performed on thirteen embalmed cadaveric specimens are also included. The osteotomy was able to expose 3.16 cm (SD = 0.60) of the subclavian vein and both the fracture and osteotomy site of all clavicles (100%) were able to be reduced and fixed using a single pre-contoured fifteen-hole lateral plate intended for use on the contralateral shoulder. This surgical technique study confirms that in the rare circumstance that the osteotomy is utilized, adequate exposure of the subclavian vein is achieved.

Tbx5 drives aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA)-signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing cardiopulmonary evolution and birth defects.