bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unravelling the Role of the Vasculature and Extracellular Matrix in Intramembranous Bone Development
Authors: Franz‐Odendaal Tamara Anne , Hammer Christine 
Published: November 2023

The relationship between bone development and the developing vasculature is well understood for endochondral bones but not for intramembranous bones. Intramembranous bones develop without a cartilage precursor from an aggregation of preosteogenic cells. Here we study, a series of intramembranous bones, known as the scleral ossicles, in order to unravel these relationships. The scleral ossicles are induced by specialised placodes of the conjunctival epithelium. We injected hydrocortisone into the developing embryo and observed the effect on the scleral bones and on the epithelial placodes. We show that there is a greater effect on the scleral bones than on the placodes, the structures that induce them. To further explore this effect, we next analysed the effect of the treatment on the developing scleral vasculature and scleral extracellular matrix. We show that both are affected indicating that vasculature and extracellular matrix within the sclera make a critical contribution to intramembranous bone development. This research provides important insights into understanding the role of the scleral mesenchyme in supporting scleral ossicle development and contributes to our understanding of sclerotic ring loss over vertebrate evolution.Support or Funding InformationNatural Sciences and Engineering Research Council, Canada
Factors Associated with Developmental Instability in the Dental Arches of Children
Authors: Miller Steven F. , Moreno Uribe Lina M. , Bay Camden , Broffitt Barbara , Levy Steven M. 
Published: November 2023

Dental arch asymmetry is often associated with malocclusion and is implicated in numerous functional deficiencies including speaking and mastication. While the existence of asymmetric dental arch shape has been well documented in malocclusion, less is known about the potential factors tied to its expression. Developmental instability refers to the suite of epigenetic factors that can disrupt the normal growth pattern of an individual and cause an increase in asymmetry. In this study, we examine the relationship of numerous health and dietary factors with developmental instability in the dental arches of children.A sample of 406 dental casts of children at age 5 from the Iowa Fluoride Study were digitized with 22 landmarks along the gingival papillae and then submitted to a Procrustes ANOVA to quantify dental arch Fluctuating Asymmetry (FA). Using FA as a metric of developmental instability, we separated individuals into low and high FA groups and then ran a series of two sample T‐Tests to see if individuals with high FA had poorer dietary or health related factors when compared to individuals with low FA.Results indicate that antibiotic usage was higher on average in children in the high FA group when compared to children with low dental arch FA (p=0.037), indicating that higher instances of childhood infection and disease may result in more asymmetric dental arches. Likewise, children with lower Vitamin D consumption had increased FA compared to children with higher levels of Vitamin D in their diet (p=0.039).These results confirm that infectious disease and diet can play a role in the normal development of the dental arches. Deficits in important nutrients, like Vitamin D, as well as elevated exposure to infectious disease, can be tied to increased dental arch asymmetry. These factors should be considered not only for the general health of a growing child, but also with regard to their proper masticatory development.Support or Funding InformationSupported by a Carver Charitable Trust Grant #12‐4058 and the NIH through Grants 2 UL1 TR000442, 1T90DE023520, R01‐DE09551 and U01‐DE018903.
Contrast Enhanced Imaging as an Alternative to Dissection in Tree Shrew (Tupaiidae: Scandentia) Chewing Musculature
Authors: Kristjanson Heather L , Perry Jonathan MG 
Published: November 2023

A burgeoning technique in the field of comparative biology involves staining specimens with iodine, followed by CT scanning. Along with providing excellent 3D visualization of soft tissue by giving it a higher attenuation, iodine staining enables digital isolation of muscles and the potential for measuring individual muscle fascicles. It is well suited for visualizing muscles of mastication, especially the medial and lateral pterygoid muscles; located internally, they are often difficult to dissect in small mammals. Here, we test the reliability of this method as an alternative to traditional dissection for obtaining accurate volume and muscle fascicle length measurements. This was accomplished by digitally isolating the masticatory muscles in three tree shrew cadaver specimens that had been subjected to iodine staining. These specimens represent the largest (Tupaia tana), smallest (T. minor), and average (T. belangeri) sizes of tree shrew. All specimens were frozen from death. After fixing with 10% buffered formalin, specimens were stained in a 3% solution of I2KI (T. tana; T. minor) or 5% solution of I2KI (T. belangeri) for 14 days. The heads were microCT scanned (at resolutions of 44.5 μm, 26.5 μm, and 32.2 μm respectively), enabling their muscles to be digitally isolated. Fascicles were visible only in T. minor due to a higher resolution, and were not measurable in any of the specimens. Larger bundles of fascicles contained within connective tissue compartments were identified and used to delineate separate muscles layers (eg. superficial vs deep masseter). These results indicate that while muscle fascicles are not measureable using these scans, they may be measurable if resolution could be increased. Isolating the muscles digitally provided more accurate measures of volume as compared to traditional dissection, while also preserving their orientation. Future work will focus on using these data to digitally map tree shrew muscles of mastication onto fossil primate mandibles as an analogue for studying early primate chewing systems.Support or Funding InformationThis work was supported through an American Association of Anatomists Short‐term Visiting Scholarship to H. Kristjanson
Tracing Cell Movement in the Bladder During Regeneration After Subtotal Cystectomy
Authors: Dettman Robert W , Devine Megan , Delos Santos Grace B , Wetterlin Jessica J , Kukulka Natalie A , Firmiss Paula R , Bowen Diana K , Gong Edward M 
Published: November 2023

Introduction and ObjectivesIt has been shown that the bladder has significant regenerative capabilities. In accordance with previous studies, there are differences between these regenerated organs and their original, healthy counterparts. The objective of this study was to determine the bladder regeneration pattern in mice with bladder resections.Methods50% of the bladder was removed in a subtotal cystectomy (STC), performed on 6–10 week female mice. DiI, a red dye, was applied to the suture site to mark the incision location. EdU was injected into STC and sham surgery mice 3 days post‐op. 4 days after injecting the EdU, Thymidine was injected to chase the remaining EdU from the mice. Bladders were fixed in blocks, cryosectioned and stained for analysis at 1, 4, and 8‐week time points. Images were taken on the Zeiss 880 Confocal microscope at 10× objective and tiled together. Using the tiled images, EdU pixel density was analyzed in Adobe Photoshop CS5. The bladders were divided into four cross‐sections: the suture site, both areas adjacent to the suture site, and bladder neck. Within each cross‐section, EdU labeled cells (yellow pixels) were counted and divided by each section's total number of cells (DAPI pixels) to determine the density of EdU per region.ResultsIt was observed that the sham bladder had EdU evenly distributed throughout the bladder. STC bladders originally had most of the EdU in the suture site, however by 4 weeks post‐op, there was a decrease in EdU at the suture site and an increase in the middle and neck areas. Overall EdU decreases over time as it gets metabolized.ConclusionsWe conclude that cell division is a response to STC. Within the 8wk study, labeled cells are migrating with a net movement away from the incision towards the bladder neck.Support or Funding InformationResearch Support Source: The Hartwell Foundation to EM Gong
Can androgens have significant effects on mesenchymal stem cells?
Authors: Firmiss Paula R , Bowen Diana K , Kukulka Natalie A , Gong Edward M , Dettman Robert 
Published: November 2023

Introduction and ObjectivesWe have identified a mesenchymal stem cell (MSC) population in the urinary bladder. In a model of fibrosis secondary to partial bladder outlet obstruction (PO) we observed that fibrosis was not as severe in female mice as compared with male mice. We hypothesized that male androgen may function in the bladder in response to obstruction.Methods8–10wk male CD‐1 mice were surgically obstructed at the bladder neck to create a partial obstruction model (PO), with and without surgical castration (CPO). Fluorescence‐activated cell sorting (FACS) was utilized to isolate MSCs (Sca‐1+/CD34+/lin− (PECAM−, CD45−, Ter119−)) in the sham, PO, and CPO bladders after 7 days of obstruction. BSCs were cultured for 24 hours and expression of androgen receptor (AR) and NKX3.1 on the BSCs was determined with immunofluorescence. Quantitative PCR was performed to determine levels of gene expression. Testosterone was replaced in castrated mice with the administration of testosterone or dihydrotestosterone.ResultsBladder function was decreased and fibrosis increased after PO. Function was normal and fibrosis reduced in CPO. Administration of testosterone or dihydrotestosterone to PO mice resulted in reduced function and increased fibrosis. The bladder MSC population was significantly decreased by PO. Castration restored this population as determined by FACS (Figure 1C). MSCs were found to express AR and twice as many sorted MSCs expressed AR (80%) by immunofluorescence after obstruction when compared to sham (40%) (Figure 1A). Microarray analysis identified the transcription factor NKX3.1 as an androgen response gene increased after PO. This was validated by qPCR and immunofluorescence staining of MSCs (Figure 1B).ConclusionsOur data suggest that testosterone directly effects bladder resident MSCs through AR and the AR target gene NKX3.1. NKX3.1 is a transcription factor that was previously thought to be expressed exclusively in the prostate gland. In fact, NKX3.1 is proposed to be expressed in and perform important functions in epithelial progenitor cells in the prostate. Activation of NKX3.1 in MSCs after obstruction may identify a maladaptive mechanism occurring in these cells that leads to fibrosis. We are currently studying a knockout model of NKX3.1 to test these ideas.Support or Funding InformationSource of Funding: The Hartwell Foundation award to EM Gong
Mesodermal differentiation of human pluripotent stem cells increases Mitofusin‐2 mediated Mitophagy
Authors: Krantz Sarah , Wang Li , Toth Peter , Marsboom Glenn , Rehman Jalees 
Published: November 2023

Pluripotent stem cells rely on glycolysis for their energy needs and shift their metabolism towards mitochondrial oxidative phosphorylation during differentiation in order to adapt to the metabolic needs of mature differentiated cells. Oxidative phosphorylation generates reactive oxygen species and can thus lead to collateral protein oxidation and damage. Excessive mitochondrial damage leads to mitochondrial autophagy or mitophagy during which mitochondria are marked for degradation. A key regulator of mitophagy is the outer mitochondrial membrane protein Mitofusin‐2 (Mfn2) which either directs mitochondria towards fusion with other mitochondria to optimize mitochondrial function or towards mitophagy. We hypothesized that differentiation of pluripotent stem cells would necessitate increased mitophagy to contain the oxidative damage due to enhanced oxidative phosphorylation.Human induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) were treated with Bone Morphogenetic Protein 4 (BMP4) to induce differentiation to the mesodermal lineage. These cells exhibited an expected increase in the mesodermal transcription factor Brachyury. Cells were stained with MitoTracker to visualize mitochondria and assess mitochondrial network formation. We observed an increase in mitochondrial fusion during mesodermal differentiation. There was also a two‐fold increase in Mitofusin 2 protein levels with BMP4 treatment. The ratiometric fluorescent sensor mitoKeima which detects the pH of mitochondria and assesses mitophagy indicated significantly higher basal mitophagy after three days of BMP4 induced differentiation with an increase in the relative mitophagy index from 2.7 to 9.7 (p<0.01) within three days.These findings suggest that mitophagy and mitochondrial networking are critical Mitofusin‐2 mediated adaptive processes required for the metabolic reprogramming of differentiating stem cells.Support or Funding InformationNIH T32 Training Grant
Finding Closure: Regulated Irf6 Expression is Required for Proper Wound Healing In Vivo
Authors: Reeb Tanner , Canady Franklin J , Tesdahl Brennan , Biggs Leah C , Dunnwald Martine 
Published: November 2023

Chronic wounds affect 6.5 million people in the US, yet little is known about the genetic and molecular mechanisms regulating wound healing. Wound closure requires the concerted action of cellular proliferation, differentiation, and migration, all processes Interferon Regulatory Factor 6 (IRF6) has been shown to regulate in keratinocytes. Embryos deficient for Irf6 demonstrated impaired ex‐vivo embryonic wound closure, and Irf6‐deficient keratinocytes displayed decreased cellular migration resulting in delayed closure in an in vitro scratch assay. Furthermore, individuals with IRF6 mutations exhibited increased likelihood of wound healing complications following surgical repair. Therefore, we hypothesize that altered expression of Irf6 will lead to impaired wound healing in adult mice. Since Irf6−/− mice are perinatal lethal, we utilized two Irf6 mutant lines: an Irf6+/− line with decreased Irf6 expression, and a mouse line with ectopic expression of the Irf6 gene under the control of a keratin 14 promoter (K14‐Irf6). K14‐Irf6 leads to overexpression of Irf6 in the basal layer of the epidermis. The adult mice were wounded with a 6 mm biopsy punch, and the wounds were assessed at either 4 or 7 days post‐wounding. Following morphometric analysis, our results indicate that at day 4, Irf6+/− mice displayed an increase in epidermal area, epidermal volume, and wound volume compared to the wild type and K14‐Irf6 mice. By day 7 the K14‐Irf6 displayed a larger wound area and wound volume as well as impaired epidermal closure compared to wild type and Irf6+/− mice, and Irf6+/− displayed a larger percentage of epidermis in the wound compared to wild type and K14‐Irf6. To determine if changes in epidermal volume could be partially attributed to changes in keratinocyte proliferation, immunohistochemistry for PCNA was performed. Our results show an increase in proliferation in the basal keratinocytes of the migratory tongue in Irf6+/− mice compared with the two other groups (P = 0.06). We also performed Masson's Trichrome staining to determine levels of granulation tissue organization. While very little collagen deposition was present 4 days post‐wounding across the three groups, we observed a trend toward decreased collagen staining and granulation tissue organization in Irf6+/− samples compared with wild type and K14‐Irf6 samples. Together, these results demonstrate that the perturbation of the levels of Irf6 (either positively or negatively) leads to defects throughout the entire period of wound healing. The morphometric analysis data suggests that underexpression of Irf6 being more detrimental in early stages of wound healing (day 4) and overexpression being more detrimental in late stages of wound healing (day 7). Ultimately, these data will provide molecular mechanisms for the increased wound healing complications observed in patients with IRF6 mutations.Support or Funding InformationR01 AR067739 (NIH)
Collagen analysis of the Achilles tendon injured and treated with F1 protein. A study using transmission electron, polarized light microscopy and western blot.
Authors: Cury Diego Pulzatto , Watanabe Ii‐Sei 
Published: November 2023

IntroductionIt is estimated that $30 billion are spent on musculoskeletal injuries in the United States each year and tendon/ligament injuries represent ~45% of these cases. Incidence of rupture of the Achilles tendon in North America is 5.5 to 9.9/100,000 habitants. After surgery, the patients return to their activity on approximately 85 days, but may range up to 270 days.AimThe effect of F1 protein extracted from Hevea brasiliensis in Achilles tendon repair in rats subjected to tenotomy.MethodsThree months‐old male Sprague Dawley rats were divided into 3 experimental groups (90 total n): control (C), Kessler‐Tajima suture (K), Kessler‐Tajima plus F1 protein (KP). Catgut suture 5‐0 was used to surgery. Transmission electron microscopy was used to analyze collagen morphology and ultrastructural aspects; western blotting was used to quantify collagen type I and III and light microscopy under polarized light to demonstrates the collagen distribution. All groups were analyzed 2 and 4 weeks after surgery.ResultsTransmission electron microscopy analysis revealed ultrastructural characteristics, in which the presence of macrophages and mast cell were noted in K and KP groups in both analyzed times. Blood vessels among collagen fibrils were noted especially in the groups analyzed after 4 weeks of injury and surgery independent of the treatment. After 2 weeks of surgery in the treated (KP) and non‐treated (K) groups the tendons' weight was increased ~7 times and decreased to ~5.6 after 4 weeks in both groups. Tendon of K group (2 weeks after injury) presented a higher ratio of collagen type III than type I when compared to all other groups, however treated groups analyzed after 2 and 4 weeks presented more collagen type I than non‐treated groups, as well as a higher density of the fibrils collagen. Polarized light microscopy shows collagen fibers arrangement for all tissue. K and KP groups (2 weeks) presented more collagen type III near muscle, and type I near of calcaneus bone; both groups after 4 weeks presented the two types of collagen along the tissue with predominance of type I. C group had only collagen type I in this type of analyze.ConclusionF1 protein increases collagen type I and density of collagen fibrils when compared to non‐treated group, therefore apparently may improve tendon repair.Support or Funding InformationFAPESP #2015/24087‐3
The History Dependence of the EMG‐Force Relationship during Ankle Dorsiflexion
Authors: Paquin James , Power Geoffrey A 
Published: November 2023

The steady‐state isometric contraction following a shortening or lengthening muscle action shows distinctly different force and activation levels as compared with a purely isometric contraction at the same muscle length. This study investigated how prior active lengthening (residual force enhancement; RFE) and shortening (force depression; FD) influences the isometric electromyography (EMG)‐Force relationship at submaximal and maximal levels as compared with purely isometric (ISO) contractions. By utilizing a series of force clamps and activation clamps at 20%, 40%, 60%, 80% and 100% of maximal isometric voluntary contraction (MVC) and maximal isometric integrated EMG (iEMG), respectively, the relative differences in neuromuscular efficiency (e.g., force/iEMG) between ISO, RFE, and FD were investigated. 17 healthy young male subjects were seated on a cybex dynamometer and performed voluntary contractions of the ankle dorsiflexors. Subjects first performed an MVC to determine relative intensities, then performed randomized active lengthening and shortening contractions at 25 deg/s over a 25 deg excursion ending at 115 degs respectively. These contractions were then compared with the purely isometric contraction at 115 degs. Surface EMG was used to record from tibialis anterior and soleus. In the RFE state when compared to the ISO state, there was significantly higher torque production during 100% MVC (~7%, P<0.05) with no significant change in agonist iEMG. Submaximally, less agonist iEMG was required to achieve the same amount of force at 60% MVC (P<0.05), and approaching significance for 40% and 20% MVC (P=0.07 and P=0.06 respectively), indicative of increased neuromuscular efficiency. In the FD state compared to the ISO state it was found that there was significantly less force produced during MVC (~15%, P<0.0001) with no significant change in agonist iEMG, but an increase in antagonist iEMG (P<0.05). In the submaximal FD state there was more agonist iEMG required to achieve the same levels of force at 80%, 60%, 40%, and 20% MVC (P<0.05), accompanied by significant increases in antagonist iEMG at 60% and 40% MVC (P<0.05). From these results, it can be concluded that the history dependent states have independent EMG‐Force relationships that exhibit significantly different levels of neuromuscular efficiency than the isometric EMG‐Force relationship.Support or Funding InformationNSERC ‐ Natural Science and Engineering Research Council of Canada