Quantitative Histomorphometry Research Articles

Bone regeneration in rabbit cranial defects: 3D printed polylactic acid scaffolds gradually enriched with marine bioderived calcium phosphate

ObjectiveThis study aimed to evaluate the in vivo biocompatibility, mechanical performance and osteoconductive potential of 3D-printed polylactic acid (PLA) scaffolds enriched with marine bioderived calcium phosphate (bioCaP) for bone tissue engineering. Materials and MethodsPLA-bioCaP composite scaffolds were specifically designed for the rabbit cranial defect model by 3D printing, with a uniform distribution of open square-shaped pores and contributions in bioCaP. Physicochemical and mechanical characterization and the evaluation of biological response are presented. ResultsThe scaffolds demonstrated mechanical properties comparable to human bones, integration with the host bone, and osteoconductive behavior promoting cell ingrowth from the defect edge. Strong mineralized tissue ingrowth through the scaffolds’ pores was observed, providing notable support to the host bone. In quantitative terms, micro-CT and histomorphometry analysis post-implantation revealed no significant differences in bone regeneration across all groups. ConclusionThe 3D-printed scaffolds with perpendicular patterning, open porosity, and proposed composition displayed satisfactory mechanical properties, biocompatibility, and osteoconductive response. The scaffolds promoted bone regeneration at similar levels as the PLA. The highest contribution of bioCaP promoted a positive influence in certain histomorphometric parameters; however, it did not significantly improve their osteogenic capability. Further research is required to optimize scaffold composition and enhance their osteogenic potential. Clinical RelevanceThis study presents a significant advancement in bone tissue engineering through the development of personalized composite scaffolds for bone-related applications. The clinical implications of this research are profound, especially considering the increasing demand for functional bone regeneration technologies capable of producing cost-effective producing cost-effective customized scaffolds.

Compositional Variations in Calcium Phosphate Cement and Poly(Lactic-Co-Glycolic-Acid) Porogens Do Not Affect the Orthotopic Performance of Calcium Phosphate Cement/Poly(Lactic-Co-Glycolic-Acid) Cements.

Calcium phosphate cement (CPC) has evolved as an appealing bone substitute material, especially since CPCs were combined with poly(lactic-co-glycolic acid) (PLGA) porogens to render the resulting CPC/PLGA composite degradable. In view of the multiple variables of CPC and PLGA used previously, the effect of CPC composition and PLGA porogen morphology (i.e., microspheres versus microparticles) on the biological performance of CPC/PLGA has not yet been investigated. Consequently, we here aimed to evaluate comparatively various CPC/PLGA formulations varying in CPC composition and PLGA porogen morphology on their performance in a rabbit femoral condyle bone defect model. CPCs with a composition of 85 wt% α-TCP, 15 wt% dicalcium phosphate anhydrate (DCPA) and 5 wt% precipitated hydroxyapatite (pHA), or 100 wt% α-TCP were combined with spherical or irregularly shaped PLGA porogens (CPC/PLGA ratio of 60:40 wt% for all formulations). All CPC/PLGA formulations were applied via injection in bone defects, as created in the femoral condyle of rabbits, and retrieved for histological evaluation after 6 and 12 weeks of implantation. Descriptive histology and quantitative histomorphometry (i.e., material degradation and new bone formation) were used for analyses. Descriptively, all CPC/PLGA formulations showed material degradation at the periphery of the cement within 6 weeks of implantation. After 12 weeks, bone formation was observed extending into the defect core, replacing the degraded CPC/PLGA material. Quantitatively, similar material degradation (up to 87%) and new bone formation (up to 28%) values were observed, irrespective of compositional variations of CPC/PLGA formulations. These data prove that neither the CPC compositions nor the PLGA porogen morphologies as used in this work affect the biological performance of CPC/PLGA formulations in a rabbit femoral condyle bone defect model.

P-011 Maternal gestational methyl donor deficiency affects reproductive development and fertility of mice offspring

Abstract Study question Is maternal methyl donor deficiency during gestation affects offspring’s reproductive health at adulthood? Summary answer Maternal gestational methyl donor deficiency affects reproductive development and fertility of F1 offspring and also leads to increased post implantation loss What is known already Reproductive disorders and their exact mechanisms are scarce in “The developmental origins of health and disease” (DOHaD) hypothesis which well explains the metabolic complications such as obesity, type 2 diabetes and hypertension that may take root during early development. The maternal nutritional environment during early development has important implications on offspring’s health later in life including reproductive health. The gestational period is of critical importance during which major epigenetic reprogramming occurs. The maternal methyl donor deficiency during gestational period may affect this process and epigenetic signatures of developing gametes and the fertility potential Study design, size, duration Pregnant female OCT4-GFP mice, fed on control chow diet (CCD) & 40% methyl-donors (Folate, Methionine & Vitamin B12) deficient diet (MDD) from gestation day (GD) 5 to GD 20. The gonadal development of F1 offsprings was studied on gestation day GD 18, post-natal day (PND) 22 and PND 60. Fertility study was done by mating adult F1 male offspring with adult female mice fed on standard diet Participants/materials, setting, methods Homocysteine levels in dams (F0) at GD18 were estimated by ELISA. Testosterone levels in F1 offsprings at PND 60 were estimated by ELISA. Furthermore, Serum glucose levels in F1 offsprings were measured at PND 60 and PND 75 by glucometer. Testicular histology, quantitative histomorphometry, immunofluorescence and TUNEL assay was performed at PND 60. Main results and the role of chance Fetal weight was reduced at GD13 and GD18 but postnatal body weight at PND 22 and 60 was not significantly affected in MDD group. Teratological defects were observed in pups of 40% MDD group as compared to CDD. Testosterone levels were significantly reduced in F1 offsprings of 40% MDD group. Glucose levels were similar in both the groups at PND 60 but at PND 75 it is significantly higher in case of F1 offsprings of 40% MDD. Maternal serum homocysteine levels and global DNA methylation levels of fetal gonads were affected. Testicular histology was significantly impaired in 40% MDD group at GD18, PND22 and PND60. Quantitative histomorphometry and immunofluorescence study showed the loss of spermatogonia and spermatocytes with significant increase in germ cells sloughing and seminiferous tubules atrophy. Apoptosis is also observed in seminiferous tubules. Copulation index, sperm count and motility was significantly reduced in case of 40% MDD group at adulthood. The F1 offspring of this group were sub-fertile with significant post implantation loss. Limitations, reasons for caution Although mice share ∼90% genome with humans, they may not resemble all aspect of physiology or pathology in humans hence it could limit the findings to extrapolate to humans. Wider implications of the findings The knowledge gained from this work could be helpful in highlighting the need of gestational micronutrient interventions specially methyl donors, which could help improving maternal and child health as well as reducing the future disease burden of reproductive disorders Trial registration number not applicable

Skeletal abnormalities in mice with Dnmt3a missense mutations

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.

Quantitative Histomorphometrical Analysis in Distinguishing Non-Hodgkin Malignant Lymphoma, Large Cell Type with Nasopharyngeal Carcinoma

Background Non-Hodgkin’s Malignant Lymphomas (NHMLs) with large cell morphology have similar histopathological appearance with Nasopharyngeal Carcinoma, undifferentiated type therefore ancillary examination such as Immunohistochemistry (IHC) are required. Quantitative histomorphometrical analysis able to identify subtle detail. The aim of this study is to measure the accuracy of quantitative histomorphometrical analysis to distinguishing NHML, large cell type with Nasopharyngeal Carcinoma. Methods Up to 33 Hematoxylin and Eosin (HE) slides from NHML, large cell type, and 27 cases of Nasopharyngeal Carcinoma were photographed under 400 times magnification with a total of 7131 images. Background, nuclei, and extra-nuclear components between cells are segmented as mathematical objects. Objects’ properties were measured and represented as distribution factors (minimum, quartile 1, median, quartile 3, maximum, range, and interquartile range). Significant factors that can differentiate the study groups are measured with a fisher-exact statistical test. Models to predict NHML, large cell type from nasopharyngeal carcinoma were formulated with different numbers of factors. The HE diagnosis accuracy compared to IHC was measured Results A sum of 7131 with 3893 from NHMLs, large cell type, and 3238 images of nasopharyngeal carcinoma were collected. After features selection, up to 76 properties factors are selected. Models' accuracy are ranging from 72.67% with a single factor and up to 89.9% with the whole selected factors. Summary Models’ accuracy are higher (72.6-89.9%) compared to HE primary diagnosis (60%) in distinguishing NHML, large cell againts nasopharyngeal carcinoma. Quantitative histomorphometry can assist pathologist for early screening to distinguishing NHML, large cell type with Nasopharyngeal Carcinoma.

Mechanical epilation exerts complex biological effects on human hair follicles and perifollicular skin: An exvivo study approach.

Electrical epilation of unwanted hair is a widely used hair removal method, but it is largely unknown how this affects the biology of human hair follicles (HF) and perifollicular skin. Here, we have begun to explore how mechanical epilation changes selected key biological read-out parameters exvivo within and around the pilosebaceous unit. Human full-thickness scalp skin samples were epilated exvivo using an electro-mechanical device, organ-cultured for up to 6 days in serum-free, supplemented medium, and assessed at different time points by quantitative (immuno-)histomorphometry for selected relevant read-out parameters in epilated and sham-epilated control samples. Epilation removed most of the hair shafts, often together with fragments of the outer and inner root sheath and hair matrix. This was associated with persistent focal thinning of the HF basal membrane, decreased melanin content of the residual HF epithelium, and increased HF keratinocyte apoptosis, including in the bulge, yet without affecting the number of cytokeratin 15+ HF epithelial stem cells. Sebocyte apoptosis in the peripheral zone was increased, albeit without visibly altering sebum production. Epilation transiently perturbed HF immune privilege, and increased the expression of ICAM-1 in the bulge and bulb mesenchyme, and the number of perifollicular MHC class II+ cells as well as mast cells around the distal epithelium and promoted mast cell degranulation around the suprabulbar and bulbar area. Moreover, compared to controls, several key players of neurogenic skin inflammation, itch, and/or thermosensation (TRPV1, TRPA1, NGF, and NKR1) were differentially expressed in post-epilation skin. These data generated in denervated, organ-cultured human scalp skin demonstrate that epilation-induced mechanical HF trauma elicits surprisingly complex biological responses. These may contribute to the delayed re-growth of thinner and lighter hair shafts post-epilation and temporary post-epilation discomfort. Our findings also provide pointers regarding the development of topically applicable agents that minimize undesirable sequelae of epilation.

'Speed-ageing' of human skin in serum-free organ culture exvivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected exvivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

FRI378 Estetrol Prolongs Anagen In Healthy Female Scalp Hair Follicles By Positively Modulating Dermal Papilla Functions And Generation Of Progenitor Stem Cells Ex Vivo

Abstract Disclosure: C. Gerard: None. A. La Riche: None. S. Altendorf: None. V. Dion: None. L. Epping: None. T. Bíro: None. G. Dixon: None. M. Bertolini: None. The human hair follicle (HF) is a neuroendocrine mini-organ that responds to, but also generates, a wide range of hormones, which affect hair cycle and HF biology. HF functions are profoundly regulated by several intrafollicular neuroendocrine axes like the HF-equivalent of the hypothalamic-pituitary-adrenal and thyroid axes, as well as by local prolactin secretion. In this regard, estrogens exert a direct role in hair cycle regulation, since estrogen receptors are prominently expressed in the epithelium of scalp HFs. It has been shown that postmenopausal women increasingly experience female pattern hair loss (FPHL) due to the decrease in ovarian estrogen secretion. Conversely, elevated estrogen levels were found to correlate with enhanced numbers of anagen HFs during pregnancy. Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. E4 was recently approved as the estrogenic component of a new combined oral contraceptive and is currently in the late stage of clinical development for its use as a menopausal hormone therapy. In this study, we aimed to assess the capacity of E4 to regulate hair growth.Frontotemporal, full-length microdissected HFs from 4 healthy female donors (> 50 years) were exposed to different concentrations of E4 (300nM, 3µM and 30µM) ex vivo. Subsequently, hair cycle staging, hair matrix keratinocyte proliferation (Ki-67), dermal papilla (DP) inductivity (versican expression and alkaline phosphatase activity), fibroblast emigration (cell density in the DP, the dermal cup (DC) and the DP stalk), and the percentage of HFs stem cell (HFSC) progenitors (CD34+) was assessed by quantitative (immuno-)histomorphometry. E4 treatment led to a tendential (300nM and 30µM) or significant (3µM) increase in the number of anagen (growing) HFs, further supported by enhanced hair matrix keratinocyte proliferation. Although neither size nor cell density of DP was affected by the treatment with E4, a significant lower number of cells was detected in the DP stalk and DC, indicating less DP fibroblast emigration. Additionally, E4 treatment stimulated DP fibroblast inductivity as evidenced by a tendential up-regulation of versican expression and alkaline phosphatase activity in the DP, the latter even significantly in only anagen HFs. Moreover, application of 3µM E4 positively affected the generation of stem cell progeny by significantly increasing the percentage of CD34+ cells in the suprabulbar outer root sheath. Taken together, our data suggest that treatment with E4 boosts anagen maintenance, reduces DP fibroblast emigration, stimulates DP inductivity and promotes the expansion of HFSC progeny. Thus, these findings point at a potential role of E4 to prevent catagen development and changes known to be involved in HF miniaturization, and further encourage the exploration of E4 as possible treatment strategy for hair loss disorders such as FPHL. Presentation: Friday, June 16, 2023

Hydroxyapatite 3D-printed scaffolds with Gyroid-Triply periodic minimal surface (TPMS) porous structure: Fabrication and an in vivo pilot study in sheep

Bone repair is a major challenge in regenerative medicine, e.g. for large defects. There is a need for bioactive, highly percolating bone substitutes favoring bone ingrowth and tissue healing. Here, a modern 3D printing approach (VAT photopolymerization) was exploited to fabricate hydroxyapatite (HA) scaffolds with a Gyroid-“Triply periodic minimal surface” (TPMS) porous structure (65% porosity, 90.5% HA densification) inspired from trabecular bone. Percolation and absorption capacities were analyzed in gaseous and liquid conditions. Mechanical properties relevant to guided bone regeneration in non-load bearing sites, as for maxillofacial contour reconstruction, were evidenced from 3-point bending tests and macrospherical indentation. Scaffolds were implanted in a clinically-relevant large animal model (sheep femur), over 6 months, enabling thorough analyses at short (4 weeks) and long (26 weeks) time points. In vivo performances were systematically compared to the bovine bone-derived Bio-OssⓇ standard. The local tissue response was examined thoroughly by semi-quantitative histopathology. Results demonstrated the absence of toxicity. Bone healing was assessed by bone dynamics analysis through epifluorescence using various fluorochromes and quantitative histomorphometry. Performant bone regeneration was evidenced with similar overall performances to the control, although the Gyroid biomaterial slightly outperformed Bio-OssⓇ at early healing time in terms of osteointegration and appositional mineralization. This work is considered a pilot study on the in vivo evaluation of TPMS-based 3D porous scaffolds in a large animal model, for an extended period of time, and in comparison to a clinical standard. Our results confirm the relevance of such scaffolds for bone regeneration in view of clinical practice. Statement of significanceBone repair, e.g. for large bone defects or patients with defective vascularization is still a major challenge.Highly percolating TPMS porous structures have recently emerged, but no in vivo data were reported on a large animal model of clinical relevance and comparing to an international standard.Here, we fabricated TPMS scaffolds of HA, determined their chemical, percolation and mechanical features, and ran an in-depth pilot study in the sheep with a systematic comparison to the Bio-OssⓇ reference.Our results clearly show the high bone-forming capability of such scaffolds, with outcomes even better than Bio-OssⓇ at short implantation time. This preclinical work provides quantitative data validating the relevance of such TMPS porous scaffolds for bone regeneration in view of clinical evaluation.

Correlation Between Testis Weight, Testis Volume, and Total Sperm Count in the Indonesian Domestic Cats (Felis catus)

Information regarding the domestic cat as a model animal in the biomedical research has not been widely carried out. This study was aimed to see the correlation between testicular weight, testicular volume and total sperm count between the left testicle, right testicle and both testes. In addition, this study was aimed to examine the effect of testicular weight and volume on quantitative histomorphometry of the testes in domestic cats in Indonesia. The results showed that there was no difference between the left and right testes. Testes weight has a positive and highly significant correlation with testes volume and total sperm count, both in the left, right, and both testes. The heavier the weight of the testes, the wider the diameter of the seminiferous tubules, the wider the lumen, and the taller the tubular epithelium. It can be concluded that the increase in testicular weight is in line with the increase in cells in the seminiferous tubules

Mouse Models of Mineral Bone Disorders Associated with Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) inevitably develop mineral and bone disorders (CKD-MBD), which negatively impact their survival and quality of life. For a better understanding of underlying pathophysiology and identification of novel therapeutic approaches, mouse models are essential. CKD can be induced by surgical reduction of a functional kidney mass, by nephrotoxic compounds and by genetic engineering specifically interfering with kidney development. These models develop a large range of bone diseases, recapitulating different types of human CKD-MBD and associated sequelae, including vascular calcifications. Bones are usually studied by quantitative histomorphometry, immunohistochemistry and micro-CT, but alternative strategies have emerged, such as longitudinal in vivo osteoblast activity quantification by tracer scintigraphy. The results gained from the CKD-MBD mouse models are consistent with clinical observations and have provided significant knowledge on specific pathomechanisms, bone properties and potential novel therapeutic strategies. This review discusses available mouse models to study bone disease in CKD.

Quantitative Histomorphometry of Pre-ovulatory Follicles and Uterine Glands of Ongole-grade Heifer in Response to the Low Doses of PMSG Administration

Injection of female mammalian animals with gonadotropin prior to mating using superovulation dose increases estradiol 17beta (E2) synthesis and secretion and improves the growth and development of the uterus to support prenatal growth with the side effect of increasing litter size. This experiment was designed to study the effects of low doses of PMSG injection in heifers on the growth of granulose cells (GC) of pre-ovulatory follicles, E2 synthesis, and uterine glands growth and development. Nine Ongole-grade heifers were divided into three groups of doses of PMSG injection, i.e., 0 IU/kg BW, 0.5 IU/kg BW, and 1.0 IU/kg BW. The injection of pregnant mare serum gonadotropin (PMSG) was conducted at the early stage of the second follicle wave. Blood samples were collected at estrus (day 0) to measure plasma E2 concentrations. The ovary and uterus were collected for histological observation after slaughtering the animal. The results showed that non-superovulation doses of PMSG injections at the second follicle wave significantly increased the growth of GC that eventually produced a significantly higher plasma E2 concentration by 39.47% and 113.57%, in heifers receiving 0.5 IU/kg BW and 1.0 IU/kg BW (p<0.05), respectively. The increase in E2 synthesis and secretion significantly stimulated the growth and development of uterine glands (p<0.05), which was reflected in the improved measures of uterine glands. In conclusion, injections of heifers with low doses of PMSG at the second follicle wave could increase the growth of GC to produce a higher secretion of E2 that eventually improves the growth and development of uterine glands in the endometrium without the risk of multiple calving.

581 Spontaneous, Intrinsic Aging in Organ-Cultured Human Skin as Novel Ex Vivo Assay to Test Candidate Anti-Aging Actives

Since in vitro methods (2D and 3D) used for the preclinical testing of candidate anti-aging compounds mimic the complexity of human skin aging unsatisfactorily, we wished to develop an ex vivo assay for interrogating key aging-read outs in healthy human skin. Healthy, full-thickness skin biopsies (2 males, 4x females; 22 - 77 years) were organ-cultured in serum-free, supplemented medium for 3 days in the presence/absence of recognized anti-aging compounds (tretinoin (5μM), resveratrol (10μM), or caffeine (200μM), and key skin aging biomarkers were analyzed by quantitative (immuno-)histomorphometry and/or qRT-PCR.

Angiotensin II Modulates Calcium/Phosphate Excretion in Experimental Model of Hypertension: Focus on Bone.

A link between hypertension and long-term bone health has been suggested. The aim of this study was to investigate the effects of chronic angiotensin II administration on urinary calcium/phosphate excretion, bone mineral density, bone remodeling and osteoblast population in a well-established experimental model of hypertension, in the absence of possible confounding factors that could affect bone metabolism. Male Sprague-Dawley rats, divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis, n = 8); (b) Ang II+losartan (Los, 50 mg/kg/day, per os, n = 6); (c) control group (physiological saline, sub cutis, n = 9); and (d) control+losartan (n = 6) were treated for four weeks. During the experimental period, 24-hour diuresis, urinary calcium, phosphate and sodium excretion were measured prior to the treatment, at two weeks of treatment, and at the end of the treatment. Systolic blood pressure was measured by plethysmography technique (tail cuff method). At the end of the experimental protocol, the rats were euthanized and peripheral quantitative computed tomography at the proximal metaphysis and at the diaphysis of the tibiae and quantitative bone histomorphometry on distal femora were performed. Angiotensin II-dependent hypertension is associated with increased calcium and phosphate excretion. AT1 receptor blockade prevented the increase of blood pressure and phosphate excretion but did not affect the increase of calcium excretion. These changes took place without significantly affecting bone density, bone histology or osteoblast population. In conclusion, in our experimental conditions, angiotensin II-dependent hypertension gave rise to an increased urinary excretion of calcium and phosphate without affecting bone density.

Human Bone Typing Using Quantitative Cone-Beam Computed Tomography

IntroductionBone typing is crucial to enable the choice of a suitable implant, the surgical technique, and the evaluation of the clinical outcome. Currently, bone typing is assessed subjectively by the surgeon. ObjectiveThe aim of this study is to establish an automatic quantification method to determine local bone types by the use of cone-beam computed tomography (CBCT) for an observer-independent approach. MethodsSix adult human cadaver skulls were used. The 4 generally used bone types in dental implantology and orthodontics were identified, and specific Hounsfield unit (HU) ranges (grey-scale values) were assigned to each bone type for identification by quantitative CBCT (qCBCT). The selected scanned planes were labelled by nonradiolucent markers for reidentification in the backup/cross-check evaluation methods. The selected planes were then physically removed as thick bone tissue sections for in vitro correlation measurements by qCBCT, quantitative micro–computed tomography (micro-CT), and quantitative histomorphometry. ResultsCorrelation analyses between the different bone tissue quantification methods to identify bone types based on numerical ranges of HU values revealed that the Pearson correlation coefficient of qCBCT with micro-CT and quantitative histomorphometry was R = 0.9 (P = .001) for all 4 bone types . ConclusionsWe found that qCBCT can reproducibly and objectively assess human bone types at implant sites.

Improvement of Outcome for Treatment of ‘Restenosis-prone’ Vascular Lesions? Potential Impact of the Paclitaxel dose on Late Lumen Loss in Porcine Peripheral Arteries

PurposeClinical data indicate that the drug density on drug-coated balloons (DCBs) might have a role on treatment effect and durability. The aim of the current study was to investigate inhibition of neointimal formation and potential adverse effects after treatment with a novel double-dose DCB in swine.Material and methodsA four-week study was performed in peripheral arteries of 12 domestic pigs after vessel injury and stent implantation. The novel double-dose DCB with 6-µg paclitaxel (Ptx)/mm2 balloon surface (1 × 6) was compared to a standard DCB with 3.5 µg Ptx/mm2 (3.5) and uncoated balloons (POBA). Potential adverse effects were stimulated by using three fully overlapping DCBs with 6 µg Ptx/mm2 each (3 × 6). Quantitative angiography, histomorphometry and histopathological analyses were performed.ResultsHigher paclitaxel doses per square millimeter of treated arteries were associated with reduced late lumen loss (LLL) in quantitative angiography 4 weeks after treatment (POBA: 0.91 ± 0.75 mm; 3.5: 0.45 ± 0.53 mm; 1 × 6: 0.21 ± 0.41 mm; 3 × 6: − 0.38 ± 0.65 mm). In histomorphometry, maximal neointimal thickness and neointimal area were the lowest for the 1 × 6 group (0.15 ± 0.06 mm/1.5 ± 0.4 mm2), followed by 3 × 6 (0.20 ± 0.07 mm/1.8 ± 0.4 mm2), 3.5 (0.22 ± 0.12 mm/2.2 ± 1.1 mm2) and POBA (0.30 ± 0.07 mm/3.2 ± 0.7 mm2). Downstream tissue showed histopathological changes in all groups including POBA, in larger number and different quality (e.g., edema, inflammation, vessel wall necrosis, vasculitis and perivasculitis) in the 3 × 6 group, which did not cause clinical or functional abnormalities throughout the study.ConclusionTreatment with the double-dose DCB (6 µg Ptx/mm2) tended to increase inhibition of in-stent neointimal formation and to diminish LLL after peripheral intervention in the porcine model compared to a market-approved DCB with 3.5 µg Ptx/mm2.

Impaired sweating in patients with cholinergic urticaria is linked to low expression of acetylcholine receptor CHRM3 and acetylcholine esterase in sweat glands.

BackgroundCholinergic urticaria (CholU), a frequent form of chronic inducible urticaria, is characterized by itchy wheals and angioedema in response to sweating. As of now, the rate and pathophysiological relevance of impaired sweating in patients with CholU are ill-defined.AimTo assess in CholU patients the rate and extent of impaired sweating and its links to clinical and pathophysiological features of CholU.Patients and methodsWe assessed sweating in patients with CholU (n = 13) subjected to pulse-controlled ergometry (PCE) provocation testing. Pre- and post-PCE biopsies of lesional (L) and non-lesional (NL) skin were analyzed for the expression of acetylcholine receptor M3 (CHRM3) and acetylcholine esterase (ACh-E) by quantitative histomorphometry and compared to those of healthy control subjects (HCs). CholU patients were assessed for disease duration and severity as well as other clinical features.ResultsOf the 13 patients with CholU, 10 showed reduced sweating in response to PCE provocation, and 3 had severely reduced sweating. Reduced sweating was linked to long disease duration and high disease severity. CholU patients with impaired sweating responses showed reduced sweat gland epithelial expression of CHRM3 and ACh-E.ConclusionReduced sweating is common in CholU patients, especially in those with long-standing and severe disease, and it can be severe. Reduced expression of CHRM3 and ACh-E may be the cause or consequence of CholU in patients with impaired sweating, and this should be explored by further studies.

An extract of Leontopodium alpinum inhibits catagen development ex vivo and increases hair density in vivo.

ObjectivesHair loss and reduction in hair volume are hallmarks of hair disorders, such as telogen effluvium, or male or female pattern hair loss, and hair ageing, which can cause severe distress in both men and women. Common anti‐hair loss drugs carry some side effects; therefore, novel, safer approaches targeting milder phenotypes are highly advocated. In this context, we investigated an extract of the alpine plant Edelweiss, Leontopodium alpinum var. Helvetia, for its ability to modulate hair follicle (HF) growth ex vivo and inhibit hair loss while increasing hair regeneration in vivo.MethodsHuman amputated HFs were microdissected from three donors, two women and one man, and cultured ex vivo for 6 days. After treatment with 0.001% Edelweiss extract (EWDE), we investigated hair shaft production and anagen/catagen conversion, and measured known parameters associated with hair growth, that is hair matrix keratinocyte proliferation and apoptosis, dermal papilla inductivity, and growth factors, by quantitative (immuno)histomorphometry. To assess the anti‐hair loss potential of the alpine plant compound, we performed a randomized, placebo‐controlled human study enrolling Caucasian women and men, aged 18 to 65 years, with normal hair loss. After 5 months’ daily use of an extract containing leave‐on serum, we analysed hair density and anagen‐to‐catagen/telogen ratio by the Trichogram analysis.ResultsOur results revealed a significant prolongation in the anagen phase in HFs treated with 0.001% Edelweiss, as indicated by an increase in HFs remaining in anagen and a significant decrease in hair cycle score. In line with this effect, EWDE significantly stimulated hair matrix (HM) keratinocyte proliferation, and dermal papilla inductivity, as shown by a significant up‐regulation of versican expression and alkaline phosphatase activity, and a tendential increase in FGF7 immunoreactivity in the dermal papilla of all HFs or only anagen VI HFs. Corroborating the ex vivo results, we observed a significant increase in growing hair shaft numbers (hair density) after treatment with Edelweiss extract formulation, and a tendential up‐regulation in the anagen‐to‐catagen/telogen ratio.ConclusionsWe show here, through several lines of evidence, that the selected extract of the alpine plant Leontopodium alpinum var Helvetia (Edelweiss) inhibits premature catagen induction, possibly by stimulating dermal papilla inductivity. It is therefore worth exploiting this extract clinically as an anti‐hair loss agent, both for preventing ageing‐associated hair shedding and as an adjuvant therapy for hair loss disorders.

Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: Comparison with changes due to jump exercise.

The preventive effects of Korean red ginseng (KRG) on bone loss and microarchitectural deterioration have been extensively studied in animal models. However, few results have been reported for the effects of KRG on the trabecular microarchitecture as compared to changes resulting from physiological stimuli such as exercise load. We compared the effects of KRG and jump exercise on improvements in trabecular microarchitecture and strength of the distal femoral metaphysis in rats. Eleven-week-old male Wistar rats were divided into sedentary (CON), KRG-administered (KRG), and jump-exercised (JUM) groups. Rats were orally administered KRG extract (200 mg/kg body weight/day) once a day for 6 weeks. The jump exercise protocol comprised 10 jumps/day, 5 days/week at a jump height of 40 cm. We used microcomputed tomography to assess the microarchitecture, volumetric bone mineral density (vBMD), and fracture load as predicted by finite element analysis at the right distal femoral metaphysis. The left femur was used for the quantitative bone histomorphometry measurements. Although KRG produced significantly higher trabecular bone volume (BV/TV) than CON, BV/TV was even higher in JUM than in KRG, and differences in vBMD and fracture load were only significant between JUM and CON. In terms of trabecular microarchitecture, KRG increased trabecular number and connectivity, whereas the JUM group showed increased trabecular thickness. Bone resorption showed significant decrease by JUM and KRG group. In contrast, bone formation showed significant increase by JUM group. These data show that KRG has weak but significant positive effects on bone mass and suggest that the effects on trabecular microarchitecture differ from those of jump exercise. The effects of combined KRG and jump exercise on trabecular bone mass and strength should be investigated.

Cadaveric Transilial Bone Biopsy: An Effective and Economic Approach to Study Bone Health

INTRODUCTIONA wide spectrum of diseases and disorders compromise bone mineral density (BMD) and bone microstructure, thus increasing the risk of bone damage and fracture. Clinically, bone imaging via dual energy X‐ray absorptiometry (DXA) and qualitative computed tomography (CT) allows for non‐destructive three‐dimensional structural analysis, but translational bone and mineral science often demands methodology that is beyond the capabilities of in vivo imaging. Human subject, transilial bone biopsy is a gold standard in translational bone and mineral science, allowing for quantitative micro‐CT and histomorphometry analyses. These analyses, however, require consent to elective surgical collection of a transilial bone biopsy, a taxing process that includes recruiting and retaining human research subjects. The purpose of this study was to review and demonstrate the use of cadaveric transilial bone analyses as an effective and economic approach to studying bone health.METHODSThe NIH National Library of Medicine electronic database was used to perform a systematic literature review according to the following cadaveric, BMD, and bone microstructure keywords: (human cadaver) AND (bone) AND (mineral density) AND (micro‐CT) OR (histomorphometry). Resulting citations were analyzed for effective and economic methodology for studying bone health. Additionally, quantitative micro‐CT and histomorphometry were performed on a cadaveric transilial bone biopsy to demonstrate protocol efficacies. 1) Superficial‐lateral gluteal muscles were reflected for open field access the gluteal surface of the iliac ala. A transilial biopsy containing both the external and internal iliac cortices was collected using an 8mm trephine at the standard human subject site, approximately 2 cm posterior and inferior to the anterior‐superior iliac spine. 2) Biopsies were subjected to compact cone‐beam tomography (micro‐CT‐40, Scanco Medical AG, Bassersdorf, Switzerland). Using 16um resolution, 3D isotropic images were collected with an integration of 250 milli‐seconds. 3) Biopsies were subjected to gradual dehydration in graded ethanol and acetone and subsequently embedded in methyl methacrylate. Serial sections were obtained with an otorizedmicrotome, stained, and mounted on glass slides.RESULTSThe systematic literature review yielded seventy‐eight citations demonstrating the utilization of human cadaveric bone to analyze bone mineral density (BMD) and bone microstructure. Twelve citations were excluded due to analysis of non‐human specimens, reporting of a case study, or manuscript access limitations. Protocol efficacies were confirmed with quantification of bone volume, connectivity density, structure model index, trabecular number, trabecular thickness, and trabecular separation via quantitative micro‐CT and histomorphometry analysis of cadaveric transilial biopsies.CONCLUSIONCadaveric transilial bone biopsy analysis via quantitative micro‐CT and histomorphometry provides an effective and economic approach to studying diseases and disorders that effect bone health.